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CN107056951A - Cd86拮抗物多靶点结合蛋白 - Google Patents

Cd86拮抗物多靶点结合蛋白 Download PDF

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CN107056951A
CN107056951A CN201710294122.XA CN201710294122A CN107056951A CN 107056951 A CN107056951 A CN 107056951A CN 201710294122 A CN201710294122 A CN 201710294122A CN 107056951 A CN107056951 A CN 107056951A
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seq
fusion protein
domain
binding domain
leu
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P·A·汤普森
P·R·包姆
P·丹
J·W·布兰肯什普
S·K·纳塔拉加
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Apatefu Research And Development Co Ltd
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Abstract

本发明提供一种多特异性融合蛋白，其由CD86拮抗物结合域和作为IL‑10激动剂、HLA‑G激动剂、HGF激动剂、IL‑35激动剂、PD‑1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物的另一个结合域组成。所述多特异性融合蛋白还可包含分隔其它结构域的间插结构域。本发明还提供编码所述多特异性融合蛋白的多核苷酸、所述融合蛋白的组合物以及使用所述多特异性融合蛋白和组合物的方法。

Description

CD86拮抗物多靶点结合蛋白
相关申请的交叉引用
本申请根据35U.S.C.§119(e)要求以下美国临时专利申请的优先权：2008年10月2日提交的61/102,288、2008年10月2日提交的61/102,297、2008年10月2日提交的61/102,307、2008年10月2日提交的61/102,315、2008年10月2日提交的61/102,319、2008年10月2日提交的61/102,327、2008年10月2日提交的61/102,331、2008年10月2日提交的61/102,334和2008年10月2日提交的61/102,336，这些(9个)临时申请通过引用全文纳入本文。
关于序列表的声明
本申请的相关序列表以文本格式代替纸件副本提供，并由此通过引用纳入说明书。包含序列表的文本名称为910180_421PC_SEQUENCE_LISTING.txt。所述文本文件为705KB，创建于2009年10月2日，通过EFS-Web以电子方式提交，与说明书一并提交申请。
背景技术
技术领域
本发明总体涉及多特异性结合分子及其治疗应用领域，更具体而言，涉及由CD86拮抗物结合域和对异源靶点如IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物具有特异性的另一个结合域组成的融合蛋白，及其组合物和治疗应用。
相关领域描述
人免疫系统通常保护身体免受外来物质和病原体的损害。免疫系统保护身体的一种方式是通过产生称为T淋巴细胞或T细胞的特化细胞。T细胞和抗原呈递细胞(APC)之间的细胞间相互作用产生T细胞共刺激信号，从而引起T细胞对抗原产生应答。完整的T细胞激活需要T细胞受体(TCR)与抗原呈递细胞上存在的抗原-MHC复合物结合以及T细胞表面上的受体CD28与抗原呈递细胞特别是树突细胞上存在的CD86和/或CD80配体结合。
CD80(也称为B7-1)最初描述为人B细胞相关激活抗原，随后发现其为相关T细胞分子CD28和细胞毒性T淋巴细胞相关抗原-4(CTLA4)的受体。在后来的研究中，鉴定出称为CD86(也称为B7-0或B7-2)的CTLA4的另一个反受体。CD86在其胞外区域与CD80具有约25％的序列相同性。尽管通常认为CD80和CD86在其启动和保持CD4(+)T细胞增殖的能力方面具有功能等效性(Vasilevko等(2002)DNA Cell Biol.21:137-49)，阻断这两种分子该活性的可溶性CTLA4Ig融合蛋白的临床数据已显示出临床益处(Genovese等(2005)NEJM 353:114-1123)，一些证据表明对CD86的特异性抑制作用可能有益。例如，CD86或CD80的介入对B细胞具有不同作用。具体而言，CD80已显示提供正常B细胞和B细胞淋巴瘤的增殖和IgG分泌的负信号，而CD86则增强B细胞的活性(Suvas等(2002)J.Biol.Chem.277:7766-7775)。还有一些证据表明，CD80介入T细胞有免疫抑制作用(Lang等(2002)J.Immunol.168:3786-3792；Taylor等(2004)J.Immunol.172:34-39；Paust等(2004)PNAS 101:10398-10403)，且其可通过PD-L1(CD274)信号传导介导对活化的APC或T细胞的进一步免疫抑制作用(Butte等(2007)Immunity 27:111-122；Keir(2008)Ann.Rev.Immunol.26:677-704)。因此，缺乏CD80抑制作用下的CD86抑制作用可在自身免疫和炎性疾病以及B细胞淋巴瘤的治疗中具有益处。
CTLA4是免疫球蛋白超家族的1型跨膜糖蛋白，其主要表达于活化T细胞中，也有一些表达发现于CD4+CD25+调节T细胞(Treg)亚群中。CD86和CD80被认为是CTLA4仅有的内源配体。已显示CTLA4与CD86和CD80结合的亲和力和亲合力高于CD28(Linsley等(1991)J.Exp.Med.174:561-69；Linsley等(1994)Immunity 1:793-801)，并作为T细胞激活的负调节物发挥关键作用。具体而言，CTLA4与CD80/CD86的结合引起T细胞应答的下调以及T细胞稳态和外周耐受性的保持。认为这种情况是由CD28依赖性共刺激的拮抗作用和通过CTLA4胞质尾区的定向负信号传导造成的。CTLA4结构和功能的综述参见Teft等(2006)Annu.Rev.Immunol.24:65-97。
如上所述，有效免疫应答同时需要TCR的介入以及CD28与CD80和/或CD86的结合。缺乏CD28结合下的TCR结合导致T细胞发生凋亡或变为无反应性。此外，已显示CD28信号传导增加T细胞细胞因子的产生。具体而言，已显示CD28刺激作用使活化T细胞中的IL-2、TNFα、淋巴毒素、IFNγ和GM-CSF的产量增加5-50倍。此外，已显示即使在免疫抑制环孢菌素的存在下，CD28也会诱导淋巴因子和/或细胞因子基因表达(Thompson等(1989)Proc.Natl.Acad.Sci.USA 86:1333-1337)。还已显示CD28通过诱导上调抗凋亡BCL-XL促进T细胞存活(Alegre等(2001)Nature Rev.Immunol.1:220-228)。
通过将CTLA4的可变样胞外结构域与免疫球蛋白恒定结构域融合提供CTLA4-Ig融合蛋白来构建CTLA4的可溶性形式。已显示可溶性CTLA-4-Ig通过与CD86和CD80二者结合防止CD28依赖性共刺激，并显示其抑制T细胞共刺激，对人具有有益的免疫抑制作用(Bruce和Boyce(2007)Ann.Pharmacother.41:1153-1162)。目前采用CTLA4-Ig融合蛋白阿巴西普(abatacept)在抗TNFα治疗应答不足的情况下治疗类风湿性关节炎。然而，并非所有患者对CTLA4-Ig均有应答，持续应答需要频繁的给药，可能部分因为阻断CD28与CD86/CD80的相互作用是Treg的弱诱导物，不足以在疾病环境中阻断活化效应T细胞应答。
附图简要说明
图1显示各种蛋白质与CD80的结合，所述蛋白质包括阿巴西普、CTLA4-Ig(N2)(SEQID NO:11)和包含与IL10融合的CTLA4胞外域的多特异性交叉受体(xceptor)融合蛋白(SEQID NO:9)。
图2显示CTLA4-Ig(N2)(SEQ ID NO:11)和包含与IL10融合的CTLA4胞外域的多特异性交叉受体融合蛋白(SEQ ID NO:9)可与可溶性IL10Ra(sIL10Ra)结合。
图3和4显示包含与IL10融合的CTLA4胞外域的多特异性交叉受体融合蛋白(SEQID NO:9)可诱导PBMC中的STAT3磷酸化。
图5显示包含来自3D1和人源化FUN1单克隆抗体的抗CD86结合域的交叉受体与WIL2-S细胞上的CD86结合。
图6显示包含CD86结合域和IL10的交叉受体可同时结合细胞表面CD86和sIL10Ra。
图7显示各种不同形式的人源化抗CD86FUN1SMIP均可结合CD86。
图8显示具有将IL10与交叉受体羧基末端(BD2)连接的各种接头的CTLA4::IL10交叉受体分子可结合IL10R1-Ig。Δ-SEQ ID NO:9；◇-SEQ ID NO:171；●-SEQ ID NO:302；▲-SEQ ID NO:173。
图9显示具有将IL10与交叉受体羧基末端(BD2)连接的各种较短接头的CTLA4::IL10交叉受体分子可结合IL10R1-Ig。Δ-SEQ ID NO:171；◇-SEQ ID NO:175；●-SEQ IDNO:177；▲-SEQ ID NO:179。
图10显示若干交叉受体蛋白结合CD80。
图11显示若干交叉受体蛋白结合CD86。
图12显示若干交叉受体蛋白结合sIL10Ra。
图13显示若干交叉受体蛋白可同时结合CD80和sIL10Ra。
图14显示若干交叉受体蛋白与小鼠CD80具有交叉反应性。
图15显示若干交叉受体蛋白与小鼠CD86具有交叉反应性。
图16和图17显示若干交叉受体蛋白在MLR试验中阻断人T细胞应答。
图18-图20显示若干交叉受体蛋白在MLR试验中阻断小鼠T细胞应答。
图21和22显示包含变体IL10(含I87突变的IL10或单IL10)或变体CTLA4的若干交叉受体蛋白在MLR试验中阻断人T细胞应答。
图23显示包含变体IL10(含I87突变的IL10或单IL10)的若干交叉受体蛋白在MC/9细胞增殖试验中的免疫刺激性小于小鼠IL10。
图24显示包含变体IL10(含I87突变的IL10或单IL10)的若干交叉受体蛋白在MC/9细胞增殖试验中的免疫刺激性小于人IL10。
发明详述
本发明使得靶向抗原呈递细胞(APC)能够改变活性。例如，T细胞活性可通过提供包含优先结合CD86的第一结合域和第二结合域(异源结合域)的多特异性交叉受体融合蛋白来调节。在某些实施方式中，多特异性交叉受体融合蛋白包含第一和第二结合域、第一和第二接头以及间插结构域，其中间插结构域的一端通过接头与作为CD86结合域的第一结合域融合，另一端通过接头与作为IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物的第二结合域融合。
在某些实施方式中，所述CD86结合域是CTLA4胞外域、CD28胞外域或具有CD86特异性的免疫球蛋白可变区结合域(如scFv)(例如来自单克隆抗体3D1或FUN1)。在一些实施方式中，采用小于完整胞外域的部分。例如，可采用CTLA4域内与CD86结合并防止CD86与CD28结合的结构域。在其它实施方式中，所述IL10激动剂是IL10或其功能区。在其它实施方式中，所述HLA-G激动剂是HLA-G5、HLA-G1、HLA-G突变蛋白或其功能区；HLA-G5、HLA-G1或HLA-G突变蛋白的胞外域；或具有ILT2、ILT4或KIR2DL4特异性的免疫球蛋白可变区结合域(如scFv)。在其它实施方式中，所述异源结合域是HGF激动剂，如HGF或其亚结构域。在另一个实施方式中，所述异源结合域是IL35激动剂，如具有IL35R、IL35或其功能区特异性的免疫球蛋白可变区结合域(如scFv)。在其它实施方式中，所述LIGHT拮抗物是具有LIGHT、HVEM胞外域或其功能区特异性的免疫球蛋白可变区结合域(如scFv)。在其它实施方式中，所述PD-1激动剂是具有PD-1、PD-1配体(例如，PD-L1或PD-L2)或其功能区特异性的免疫球蛋白可变区结合域(如scFv)。在其它实施方式中，所述BTLA激动剂是具有BTLA、HVEM胞外域或其功能区特异性的免疫球蛋白可变区结合域(如scFv)。在某些实施方式中，所述GITRL拮抗物是具有GITRL、GITR胞外域、可溶性GITR或其功能区特异性的免疫球蛋白样可变区结合域(如scFv)。在某些实施方式中，CD40拮抗物是具有CD40特异性的免疫球蛋白样可变区结合域(如scFv)。
这样的多特异性融合蛋白在本文称为交叉受体(Xceptor)分子，其示例性结构包括N-BD-ID-ED-C、N-ED-ID-BD-C、N-BD1-ID-BD2-C和N-ED-ID-ED-C，其中N-和–C分别指氨基和羧基末端；BD是免疫球蛋白样或免疫球蛋白可变区结合域；ID是间插结构域；ED是细胞外或胞外域，如受体配体结合域、配体、C型凝集素结构域、导向蛋白或导向蛋白样结构域或类似结构。在一些构建体中，ID可包含置于第一和第二结合域之间的免疫球蛋白恒定区或亚区。在其它构建体中，BD和ED各自通过相同或不同的接头(例如包含1-50个氨基酸的接头)，如免疫球蛋白绞链区(由例如上部区域和核心区域构成)或其功能性变体，或凝集素域间区或其功能性变体，或分化簇(CD)分子柄区域(stalk region)或其功能性变体与ID连接。
在更详细地阐述本发明之前，提供本文所用某些术语的定义可能有助于理解本发明。其它定义在本说明书全文中列出。
在本说明书中，任何浓度范围、百分数范围、比例范围或整数范围应理解为包括所述范围内的任何整数值，适当时也包括其分数值(如整数的十分之一和百分之一)，除非另有说明。另外，本文所述的有关任何物理特征，如聚合物亚基、大小或厚度的任何数值范围应理解为包括所述范围内的任何整数，除非另有说明。本文所用的“约”或“主要由…组成”指所述范围、数值或结构的±20％，除非另有说明。应理解，本文所用术语“一个”和“一种”指“一个或多个”所列举的组分。替代物的使用(例如“或”)应理解为所述替代物中的一个、两个或其任何组合。本文所用术语“包括”和“包含”可作为同义词使用。此外，应理解本申请公开衍生自本文所述结构和取代基的各种组合的单个化合物或化合物组就好像各化合物或化合物组单独列出那样相同的程度。因此，特定结构或特定取代基的选择属于本发明范围内。
本发明所述的“结合域”或“结合区”可以是例如，具有特异性识别和结合生物分子(如CD86)或一个以上相同或不同分子的复合物或装配体或聚集体(不论它是稳定的或是瞬时的，例如CD86/CD28复合物)的能力的任何蛋白质、多肽、寡肽或肽。这类生物分子包括蛋白质、多肽、寡肽、肽、氨基酸或其衍生物；脂质、脂肪酸或其衍生物；碳水化合物、糖类或其衍生物；核苷酸、核苷、肽核酸、核酸分子或其衍生物；糖蛋白、糖肽、糖脂、脂蛋白、蛋白脂类或其衍生物；生物样品中可能存在的其它生物分子；或者它们的任何组合。结合区包括生物分子或其它感兴趣靶点的任何天然产生、合成、半合成或重组产生的结合伙伴。已知鉴定特异性结合特定靶点的本发明的结合域的各种试验，包括FACS、Western印迹、ELISA或Biacore分析。
如果本发明的结合域和其融合蛋白结合靶分子的亲和力或Ka(即特定结合相互作用的平衡结合常数，单位是1/M)例如大于或等于约105M-1、106M-1、107M-1、108M-1、109M-1、1010M-1、1011M-1、1012M-1或1013M-1，则它们能够以所需程度结合，包括“特异性或选择性结合”靶点，而不显著结合测试样品中的其它组分。“高亲和力”结合域指Ka为至少107M-1、至少108M-1、至少109M-1、至少1010M-1、至少1011M-1、至少1012M-1、至少1013M-1或更大的结合域。或者，亲合力可定义为特定结合相互作用的平衡解离常数(Kd)，单位是M(例如10-5M-10-13M)。本发明所述结合域多肽和融合蛋白的亲和力可采用常规技术很容易地测定(参见例如，Scatchard等(1949)Ann.N.Y.Acad.Sci.51:660；和美国专利号5,283,173、5,468,614或等效物)。
可如本文所述或通过本领域已知的各种方法产生本发明的结合域(参见例如，美国专利6,291,161、6,291,158)。来源包括来自各种物种，包括人、驼科动物(来自骆驼、单峰骆驼或美洲驼；Hamers-Casterman等(1993)Nature，363:446和Nguyen等(1998)J.Mol.Biol.，275:413)、鲨鱼(Roux等(1998)Proc.Nat’l.Acad.Sci.(USA)95:11804)、鱼(Nguyen等(2002)Immunogenetics，54:39)、啮齿动物、禽类、绵羊的抗体基因序列(形式可以是sFv、scFv或Fab，如噬菌体文库中的情况)，编码随机肽文库的序列或编码其它替代性非抗体支架的环区域中工程改造的多种氨基酸的序列，如血纤蛋白原结构域(参见例如，Weisel等(1985)Science 230:1388)、库尼茨(Kunitz)结构域(参见例如，美国专利6,423,498)、脂质运载蛋白结构域(参见例如，WO 2006/095164)、V样结构域(参见例如，美国专利申请公开号2007/0065431)、C-型凝集素结构域(Zelensky和Gready(2005)FEBS J.272:6179)、mAb2或FcabTM(参见例如PCT专利申请公开号WO2007/098934、WO 2006/072620)或类似序列。此外，也可采用例如将合成的单链CD86作为方便系统(例如，小鼠、HuMAbTC小鼠TM、美洲驼、鸡、大鼠、仓鼠、家兔等)中的免疫原的杂交瘤开发传统策略来开发本发明的结合域。
除非另有说明，抗体技术领域技术人员理解的术语各自具有所述领域获得的含义。例如，术语“VL”和“VH”分别指衍生自抗体轻链和重链的可变结合区。可变结合区由离散的良好限定的亚区域构成，这些区域称为“互补决定区”(CDR)和“框架区”(FR)。术语“CL”和“CH”指“免疫球蛋白恒定区”，即分别指衍生自抗体轻链或重链的恒定区，后者还理解为根据衍生该区域的抗体的同种型(IgA、IgD、IgE、IgG、IgM)进一步分为CH1、CH2、CH3和CH4恒定区结构域。恒定区结构域的一部分构成Fc区(“可结晶片段”区)，其包含负责免疫球蛋白效应物功能如ADCC(抗体依赖性细胞介导的细胞毒性)、CDC(补体依赖性细胞毒性)和补体结合、与Fc受体结合、相对于缺乏Fc区的多肽体内半衰期延长、蛋白A结合、可能甚至是胎盘转移的结构域(参见Capon等(1989)Nature，337:525)。此外，含有Fc区的多肽能够发生多肽的二聚化或多聚化。“绞链区”在本文也称为“接头”，它是插于抗体单链的可变结合区和恒定区之间并连接二者的氨基酸序列，本领域已知它以绞链形式为抗体或抗体样分子提供柔性。
免疫球蛋白的结构域结构适合工程改造，可在免疫球蛋白分子不同类和亚类之间交换抗原结合域和产生效应物功能的结构域。有关免疫球蛋白结构和功能的综述参见例如Harlow等编，《抗体：实验室手册(Antibodies:A Laboratory Manual)》，第14章(冷泉港实验室出版社(Cold Spring Harbor Laboratory)，冷泉港(Cold Spring Harbor)，1988)。有关重组抗体技术的各方面的大量介绍和详细信息可参见教科书《重组抗体》(约翰韦利森出版社(John Wiley&Sons)，纽约，1999)。有关抗体工程的详细实验方案的全面集合可参见R.Kontermann和S.Dübel编，《抗体工程实验室手册(The Antibody Engineering LabManual)》(施普林格出版社(Springer Verlag),海德堡/纽约,2000)。其它相关方案还可参见马萨诸塞州波士顿的约翰韦利森出版社出版的《当代免疫学方案(Current Protocolsin Immunology)》(2009年8月)。
本文所用的“衍生物”指化学或生物学改性形式的化合物，其结构类似于母体化合物且(实际或理论上)可从该母体化合物获得。通常，“衍生物”不同于“类似物”，母体化合物可以是产生“衍生物”的原料，但母体化合物不一定用作产生“类似物”的原料。类似物可具有与母体化合物不同的化学或物理性质。例如，与母体化合物相比，衍生物可能亲水性更高或反应性改变(例如具有改变其靶点亲和力的氨基酸改变的CDR)。
术语“生物学样品”包括来自对象或生物来源的血样、活检样品、组织外植体、器官培养物、生物流体(例如，血清、尿液、CSF)或任何其它组织或细胞或其它制备物。对象或生物来源可以是例如，人或非人动物、原代细胞培养物或适合培养的细胞系，包括可含有染色体整合的或附加体形式的重组核酸序列的遗传工程改造细胞系、体细胞杂交细胞系、永生化或可永生化细胞系、分化或可分化细胞系、转化的细胞系或类似细胞系。在本发明的其它实施方式中，对象或生物来源可疑似患有疾病、失调或病症，或处于患上疾病、失调或病症的风险中，所述疾病、失调或病症包括恶性疾病、失调或病症或B细胞疾病。在某些实施方式中，对象或生物来源可疑似患有过度增殖、炎症或自身免疫疾病或者处于发生这些疾病的风险中，在本发明某些其它实施方式中，对象或生物来源可以是已知不具患这样的疾病、失调或病症的风险，或不存在这样的疾病、失调或病症。
CD86结合域
如本文所述，CD86包含作为免疫球蛋白超家族成员的I型膜蛋白。CD86由抗原呈递细胞表达，其为两种T细胞蛋白CD28和CTLA4的配体。CD28与CD28的结合是T细胞激活作用的共刺激信号，而CD28与CTLA4的结合则下调T细胞激活作用并降低免疫应答。可变剪接产生编码不同同种型的两种转录物变体(Genbank登录号NP_787058.3和NP_008820.2)。
本发明的CD86结合域可阻断CD86结合CD28，由此下调T细胞激活作用。所考虑的CD86结合域包括CTLA4胞外结构域或其亚结构域、CD28胞外结构域或其亚结构域、CD86特异性抗体衍生结构域(如衍生自FUN1单克隆抗体(参见例如，J Pathol.1993年3月；169(3):309-15)；或衍生自3D1抗CD86单克隆抗体)。
在一些实施方式中，CD86结合域是人CTLA4(Genebank登录号NP_005205)的胞外结构域(“胞外域”)，如SEQ ID NO:1的成熟多肽序列(信号肽：氨基酸1-37)。不含信号肽的CTLA4胞外域的氨基酸序列见SEQ ID NO:410所提供。申请人注意到某些研究显示CTLA4胞外域的成熟多肽从SEQ ID NO:1第38位的甲硫氨酸开始，其它研究显示所述成熟多肽从第37位的丙氨酸开始。在其它实施方式中，CD86结合域是经突变以使对CD86的亲和力高于野生型或非突变型CTLA4的CTLA4胞外域，如例如美国专利申请公开号US 2003/0035816所公开的。在某些实施方式中，突变CTLA4胞外域在SEQ ID NO:410的第29位氨基酸包含丙氨酸或酪氨酸，和/或在第104位包含谷氨酸、天冬酰胺、天冬氨酸、谷氨酰胺、异亮氨酸、亮氨酸或苏氨酸。A29Y L104E CTLA 4胞外域变体的氨基酸序列见SEQ ID NO:411所提供。在某些实施方式中，CD86结合域是CTLA-4可变样结构域，如SEQ ID NO:3提供的序列；或CTLA-4可变样结构域的CDR，如SEQ ID NO:4(CDR1)、SEQ ID NO:5(CDR2)或SEQ ID NO:6(CDR3)。这样的CDR见例如美国专利7,405,288描述。在替代性实施方式中，CD86结合域是CD28胞外结构域(“胞外域”)(Genbank登录号NP_006130.1)，如SEQ ID NO:2提供的序列。SEQ ID NO:2的氨基酸1-18是信号肽。不含信号肽的CD28胞外域的氨基酸序列见SEQ ID NO:412所提供。
在其它实施方式中，CD86结合域包含具有CD86特异性的单链免疫球蛋白样结构域，如scFv。在某些实施方式中，所述CD86结合域包含来自单克隆抗体FUN1或3D1的轻链和重链可变结合域。来自FUN1和3D1抗CD86单克隆抗体的重链、轻链、scFv接头和CDR的序列分别见SEQ NO:305-313和318-326所示，可用于本发明的交叉受体分子。
在一方面，本发明的CD86结合域或其融合蛋白具有CD86特异性，其亲和力的离解常数(Kd)为约10-3M到小于约10-8M。在某些优选实施方式中，所述CD86结合域或其融合蛋白与CD86结合的亲和力为约0.3μM。
在一个示例性实施例中，可利用Fab片段噬菌体文库(参见例如，Hoet等(2005)Nature Biotechnol.23:344)，通过筛选与合成或重组的CD86(使用GenBank登录号NP_787058.3或NP_008820.2所示的氨基酸序列或其片段)的结合，鉴定本发明的CD86结合域。在某些实施方式中，用于产生CD86结合域的CD86分子还可包含本文所述的间插结构域或二聚化结构域，如免疫球蛋白Fc结构域或其片段。
在一些实施方式中，本发明的CD86结合域包含本文所述的VH和VL结构域(例如，FUN1、3D1或其人源化衍生物)。其它示例性VH和VL结构域包括美国专利6,827,934所述的结构域。在某些实施方式中，所述VH和VL结构域是人的结构域。在其它实施方式中，提供的本发明CD86结合域具有与SEQ NO:305和306、SEQ NO:318和319或美国专利6,827,934(通过引用纳入本文)公开序列的一种或多种轻链可变区(VL)或一种或多种重链可变区(VH)或二者的氨基酸序列至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、至少99.5％或至少100％相同的序列，其中各CDR可具有0、1、2或3个氨基酸改变(即多数改变位于框架区中)。
在两种或多种多肽或核酸分子序列中，术语“相同”或“相同性百分数”指在比较窗口或指定区域上，采用本领域已知方法如序列比较算法，通过手工比对或目测检查来比较和比对最大对应性时，两个或多个序列或子序列在指定区域相同或其中有一定百分数的氨基酸残基或核苷酸相同(例如，60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％相同)。例如，适合测定序列相同性百分数和序列相似性百分数的优选算法是BLAST和BLAST 2.0算法，分别见Altschul等(1977)NucleicAcids Res.25:3389和Altschul等(1990)J.Mol.Biol.215:403所述。
在本文所述的可能需要VL和VH结构域的任何这些或其它实施方式中，VL和VH结构域可以任何取向排列，可由本文所述的最多约30个氨基酸的接头或能够提供与两个亚结合域相互作用相容的间隔物功能的任何氨基酸序列分隔开。在某些实施方式中，连接VH和VL结构域的接头包含SEQ ID NO：43-166、244、307、320、355-379和383-398中一个或多个所示的氨基酸序列，如接头46(SEQ ID NO:88)、接头130(SEQ ID NO:163)、接头131(SEQ ID NO:164)、接头115(SEQ ID NO:148)或SEQ ID NO:244中提供的接头。多特异性结合域将具有至少两个特异性亚结合域，其类似于驼类抗体组成；或具有至少四个特异性亚结合域，其类似于成对VH和VL链的更常规的哺乳动物抗体组成。
本领域以多种方式定义了CDR，包括Kabat、Chothia、AbM和接触定义。Kabat定义基于序列可变性，是预测CDR区的最常用定义(Johnson等(2000)Nucleic Acids Res.28:214)。Chothia定义基于结构环区域的位置(Chothia等(1986)J.Mol.Biol.196:901；Chothia等(1989)Nature 342:877)。AbM定义在Kabat和Chothia定义之间作出妥协，它是牛津分子集团(Oxford Molecular Group)开发的用于抗体结构建模的完整程序包(Martin等(1989)Proc.Nat′l.Acad.Sci.(USA)86:9268；Rees等，ABMTM，“用于抗体可变区建模的计算机程序(a computer program for modeling variable regions of antibodies)”，Oxford，UK；牛津分子公司(Oxford Molecular,Ltd.))。近来引入了称为接触定义的另外一种定义(参见MacCallum等(1996)J.Mol.Biol.5:732)，该定义基于对可用复合物晶体结构的分析。
按照惯例，重链中的CDR结构域称为H1、H2和H3，它们是从氨基末端向羧基末端顺序编号的。CDR-H1长度约为10-12个残基，根据Chothia和AbM定义从Cys之后4个残基处开始，或根据Kabat定义从其后5个残基处开始。H1后可接Trp、Trp-Val、Trp-Ile或Trp-Ala。根据AbM定义，H1的长度约为10-12个残基，而Chothia定义则排除了最后四个残基。根据Kabat和AbM定义，CDR-H2从H1末端后15个残基处开始，通常其前接序列Leu-Glu-Trp-Ile-Gly(但已知多个变异)且通常后接序列Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala。根据Kabat定义，H2的长度为约16-19个残基，而AbM定义预测所述长度为9-12个残基。CDR-H3通常从H2末端后33个残基处开始，通常前接氨基酸序列Cys-Ala-Arg且后接氨基酸Gly，其长度范围是3到约25个残基。
按照惯例，轻链中的CDR区称为L1、L2和L3，它们是从氨基末端向羧基末端顺序编号的。CDR-L1(长度约为10-17个残基)通常从约残基24处开始，并通常前接Cys。CDR-L1之后的残基总是Trp，以其开始以下序列中的一种：Trp-Tyr-Gln、Trp-Leu-Gln、Trp-Phe-Gln或Trp-Tyr-Leu。CDR-L2(长度约为7个残基)从L1末端后约16个残基处开始，通常前接残基Ile-Tyr、Val-Tyr、Ile-Lys或Ile-Phe。CDR-L3通常从L2末端后33个残基处开始，通常前接Cys，且通常后接序列Phe-Gly-XXX-Gly，其长度约为7-11个残基。
因此，本发明的结合域可包含来自抗CD86抗体的可变区的单个CDR，或其可包含多个相同或不同的CDR。在某些实施方式中，本发明的结合域包含具有CD86特异性的VH和VL结构域，其包含框架区以及CDR1、CDR2和CDR3区，其中(a)所述VH结构域包含重链CDR3的氨基酸序列；或(b)所述VL结构域包含轻链CDR3的氨基酸序列；或(c)所述结合域包含(a)的VH氨基酸序列和(b)的VL氨基酸序列；或所述结合域包含(a)的VH氨基酸序列和(b)的VL氨基酸序列，且其中VH和VL发现于同一参比序列中。在其它实施方式中，本发明的结合域包含具有CD86特异性的VH和VL结构域，其包含框架区以及CDR1、CDR2和CDR3区，其中(a)所述VH结构域包含重链CDR1、CDR2和CDR3的氨基酸序列；或(b)所述VL结构域包含轻链CDR1、CDR2和CDR3的氨基酸序列；或(c)所述结合域包含(a)的VH氨基酸序列和(b)的VL氨基酸序列；或所述结合域包含(a)的VH氨基酸序列和(b)的VL氨基酸序列，其中所述VH和VL氨基酸序列来自同一参比序列。
在本文所述的包含特异性CDR的任何实施方式中，结合域可包含(i)VH结构域，其具有与VH结构域的氨基酸序列至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列；或(ii)VL结构域，其具有与VL结构域的氨基酸序列至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列；或(iii)同时包含(i)的VH结构域和(ii)的VL结构域；或同时包含(i)的VH结构域和(ii)的VL结构域，其中VH和VL来自同一参比序列，且各CDR具有最多3个氨基酸改变(即多个改变位于框架区中)。
本发明交叉受体融合蛋白中的CD86结合域可以是免疫球蛋白样结构域，如免疫球蛋白支架。本发明考虑的免疫球蛋白支架包括scFv、结构域抗体或仅含重链的抗体。在scFv中，本发明考虑到重链和轻链可变区由本文所述或本领域已知的任何接头肽连接，以与结合分子中连接的结构域或区域相容。示范性接头是基于Gly4Ser接头基序的接头，例如(Gly4Ser)n，其中n＝1-5。如果本发明融合蛋白的结合域基于非人免疫球蛋白或包含非人CDR，则所述结合域可根据本领域已知方法“人源化”。
或者，本发明融合蛋白的CD86结合域可以是除免疫球蛋白支架以外的支架。本发明考虑的其它支架能以功能性构象呈递CD86特异性CDR。所考虑的其它支架包括但不限于：A结构域分子、纤连蛋白III结构域、抗脂质运载蛋白(anticalin)，锚蛋白重复工程改造的结合分子、腺连蛋白(adnectin)，库尼茨(Kunitz)结构域或蛋白AZ结构域亲和体(affibody)。
IL10
如上所述，在某些实施方式中，本发明提供含有作为IL10激动剂(即可提高IL10信号传导)的结合区或结合域的多肽。在一些实施方式中，IL10激动剂结合域是IL10或IL10Fc，或其功能性亚结构域。在其它实施方式中，所述IL10激动剂结合域是特异性结合IL10R1或IL10R2的单链结合蛋白，如scFv。在一些实施方式中，所述IL10激动剂结合域是包含SEQ ID NO:7第87位点突变，如从“I”变为“A”或“S”(本文分别称为I87A或I87S)的IL10。已知I87变体IL10分子与野生型IL10相比免疫刺激性较小(Ding等,J.Exp.Med.191:213,2000)。此外，IL10通常形成同型二聚体，其中某一单体分子的氨基末端结构域结合于另一个单体羧基末端结构域。在一个实施方式中，所述IL10激动剂结合域是具有较短接头(gggsgg SEQ ID NO:379)的IL10分子，所述接头将所述分子的两个亚结构域(氨基和羧基末端结构域)分隔开，以使这些亚结构域可形成分子内二聚体，也对其进行检测。这些在本文称为单IL10分子。
IL10(Genbank登录号NP_000563.1；SEQ ID NO：7)是共享α-螺旋结构的细胞因子超家族的成员。SEQ ID NO:7的氨基酸1-18是前体IL10蛋白的信号肽。成熟IL10蛋白的氨基酸序列见SEQ ID NO:418所提供。虽然不存在经验证据，但据提示所有家族成员均具有6个α-螺旋(Fickenscher，H.等，(2002)Trends Immunol.23:89)。IL10含有4个半胱氨酸，其中只有一个在家族成员中具有保守性。由于IL10显示了有助于二聚化的V-形折叠，因此看来二硫键对该结构并不重要。家族成员与IL10的氨基酸相同性在20％(IL-19)到28％(IL-20)之间变化(Dumouter等，(2002)Eur.Cytokine Netw.13:5)。
IL10最初被描述为小鼠中抑制Th1细胞生产IFN-α和GM-CSF细胞因子的Th2细胞因子(Moore等，2001，Annu.Rev.Immunol.19:683；Fiorentino等(1989)J.Exp.Med.170:2081)。人IL10的长度为178个氨基酸，含有18个氨基酸的信号序列和160个氨基酸的成熟区段。其分子量为约18kDa(单体)。人IL10不含潜在的N连接糖基化位点，且不发生糖基化(Dumouter等，(2002)Eur.Cytokine Netw.13:5；Vieira等(1991)Proc.Natl.Acad.Sci.USA88:1172)。其含有4个半胱氨酸残基，形成两个链内二硫键。一个单体的螺旋A-->D与第二单体的螺旋E和F发生非共价相互作用，形成非共价的V形同型二聚体。已对IL10分子上的功能性区域作图。在N末端，前螺旋A残基#1-9参与肥大细胞增殖，而在C-末端，螺旋F残基#152-160介导白细胞分泌和趋化性。
已知表达IL10的细胞包括CD8+T细胞、小胶质细胞、CD14+(但非CD16+)单核细胞、Th2CD4+细胞(小鼠)、角质形成细胞、肝星状细胞、Th1和Th2CD4+T细胞(人)、黑色素瘤细胞、活化的巨噬细胞、NK细胞、树突细胞、B细胞(CD5+和CD19+)和嗜酸性粒细胞。现在认为，最初观察到IL10对T细胞IFN-γ产生的抑制作用是辅助细胞介导的间接作用。然而，对T细胞的其它作用包括：IL10诱导的CD8+T细胞趋化性、CD4+T细胞向IL-8的趋化性的抑制作用、激活后的IL-2产生的抑制作用、通过Bcl-2上调对T细胞凋亡的抑制作用和伴随CD28共刺激的低抗原暴露后的T细胞增殖的中断作用(Akdis等，(2001)Immunology 103:131)。
IL10对B细胞具有多个相关但独特的功能。IL10能与TNF-β和CD40L一起诱导幼稚(IgD+)B细胞产生IgA。据信，TGF-β/CD40L促进类型转换，而IL10启动分化和生长。不存在TGF-β时，IL10与CD40L协作诱导IgG1和IgG3(人)，因此可能是IgG亚型的直接转换因子。有趣的是，IL10对IL-4诱导的IgE分泌具有分歧作用。如果IL10在IL-4诱导类型转换时存在，则其能逆转所述作用；如果其在IgE定型后存在，其会增加IgE的分泌。最后，IL10存在下的CD27/CD70相互作用会促进由记忆B细胞形成浆细胞(Agematsu等(1998)Blood 91:173)。
肥大细胞和NK细胞也受IL10的影响。IL10能诱导肥大细胞释放组胺，同时阻断其释放GM-CSF和TNF-α。该作用可以是自分泌，因为已知大鼠的肥大细胞能释放IL10。作为其多效性的证据，IL10对NK细胞具有相反的作用。IL10实际上能促进NK细胞产生TNF-α和GM-CSF，而非阻断该功能。此外，它能增强IL-2诱导的NK细胞增殖并促进IL-18致敏的NK细胞分泌IFN-γ。与IL-12和/或IL-18一致，IL10能增强NK细胞的细胞毒性(Cai等，1999，Eur.J.Immunol.29:2658)。
IL10对嗜中性粒细胞有显著的抗炎作用。它能抑制趋化因子MIP-1α、MIP-1β和IL-8的分泌，并阻断促炎介导物IL-1β和TNF-α的产生。此外，它能降低嗜中性粒细胞产生超氧化物的能力，因而会干扰PMN介导的抗体依赖性细胞毒性。它也阻断IL-8和fMLP诱导的趋化性，可能通过CXCR1发挥该作用(Vicioso等，(1998)Eur.Cytokine Netw.9:247)。
IL10通常对树突细胞(DC)具有免疫抑制作用。它显示以DC为代价促进CD14+巨噬细胞分化。IL10似乎能降低DC刺激T细胞，特别是Th1型细胞的能力。相对于MHC-II表达，它可能被下调、不变或上调(Sharma等，(1999)J.Immunol.163:5020)。至于CD80和CD86，IL10能上调或下调其表达。B7-2/CD86在T细胞激活中起到关键作用。就该分子而言，IL10同时参与其上调和下调作用。然而，最显著的调节作用也许出现在CD40上(IL10显示降低其表达)。在区域水平，IL10可通过响应于促炎细胞因子抑制朗格汉斯细胞迁移而阻断免疫刺激。或者，IL10阻断炎症诱导的DC成熟步骤，所述步骤通常包括CCR1、CCR2和CCR5下调以及CCR7上调。该阻断作用连同CCR1、CCR2和CCR5的保持导致DC无法迁移到区域性淋巴结。结果是产生不会刺激T细胞但会结合(和清除)促炎趋化因子而不对其产生应答的不移动的DC(D-Amico等，(2000)Nat.Immunol.1:387)。
已报道IL10对单核细胞具有多种作用。例如，IL10显示能明显降低细胞表面MHC-II的表达。其还在刺激后抑制IL-12的产生。虽然它能与M-CSF联合促进单核细胞向巨噬细胞转变，但巨噬细胞的表型不明显(即CD16+/细胞毒性相对CD16-)。IL10也能降低单核细胞的GM-CSF分泌量和IL-8产量，同时促进IL-1ra的释放(Gesser等，(1997)Proc.Natl.Acad.Sci.USA94:14620)。透明质粘连蛋白是结缔组织的组分，已知它由单核细胞响应于IL10而分泌。这可能对细胞迁移，特别是肿瘤细胞转移具有重要影响，已知透明质粘连蛋白能破坏细胞通过胞外空间的迁移(Gesser等，(1997)Proc.Natl.Acad.Sci.USA94:14620)。
已显示IL10与鼠或猕猴Fc区的融合蛋白(称为IL10Fc)能抑制巨噬细胞功能并延长胰岛异源移植体的存活(Feng等(1999)Transplantation68:1775；Asiedu等(2007)Cytokine 40:183)，并减轻鼠模型中的败血性休克(Zheng等(1995)J.Immunol.154:5590)。
人IL10R1是90-110kDa的、I型单次跨膜糖蛋白，其表达于有限数量的细胞类型(Liu等，1994，J.Immunol.152:1821)。在胰、骨骼肌、脑、心和肾中观察到弱表达。胎盘、肺和肝显示中等水平。单核细胞、B细胞、大粒淋巴细胞和T细胞高水平表达(Liu等，1994，J.Immunol.152:1821)。表达的蛋白是578个氨基酸的蛋白，其包含21个氨基酸的信号肽、215个氨基酸的胞外区、25个氨基酸的跨膜区和317个氨基酸的胞质结构域。胞外区内存在两个FNIII基序，胞质域内存在STAT3停靠位点和JAK1结合区(Kotenko等，2000Oncogene19:2557；Kotenko等，1997，EMBO J.16:5894)。IL10R1结合人IL10的Kd为200pM。
在一些实施方式中，本发明的结合域包含如本文所述具有IL10R1或IL10R2特异性的VL和VH结构域。在某些实施方式中，所述VL和VH结构域是人的结构域。VL和VH结构域可以任何取向排列，并可由本文所述的至多约30个氨基酸的接头或能够提供与两个亚结合域相互作用相容的间隔物功能的任何其它氨基酸序列分隔开。在某些实施方式中，连接VL和VH结构域的接头包含SEQ ID NO：43-166、244、307、320、355-379和383-398中所示的氨基酸序列，如SEQ ID NO:244中提供的接头、接头46(SEQ ID NO:88)、接头130(SEQ ID NO:163)或接头131(SEQ ID NO:164)。多特异性结合域可具有至少2个特异性亚结合域，其类似于驼类抗体组成；或具有至少4个特异性亚结合域，其类似于成对VL和VH链的更常规哺乳动物抗体组成。在其它实施方式中，本发明具有IL10R1或IL10R2特异性的结合域可包含一个或多个互补决定区("CDR")，或多个拷贝的一种或多种这类CDR，这类CDR由抗-IL10R1或抗IL10R2的scFv或Fab片段可变区或其重链或轻链可变区获得、衍生或设计出。因此，本发明的结合域可包含来自抗-IL10R1或抗-IL10R2的可变区的单个CDR，或可包含多个相同或不同的CDR。在某些实施方式中，本发明的结合域包含具有IL10R1或IL10R2特异性的VL和VH结构域，其包含框架区和CDR1、CDR2和CDR3区。
HLA-G
如上所述，在某些实施方式中，本发明提供含有作为HLA-G激动剂(即可提高IL10信号传导)的结合区或结合域的多肽。在一些实施方式中，所述HLA-G激动剂结合域是HLA-G1(SEQ ID NO:14)、HLA-G5(SEQ ID NO:15)或HLA-G突变蛋白，其中成熟蛋白第147位的半胱氨酸突变为替代氨基酸例如丝氨酸。HLA-G1和HLA-G5的氨基酸1-24和1-23分别表示信号肽。在其它实施方式中，所述HLA-G激动剂结构域是HLA-G1或HLA-G5的胞外域，含或不含通过柔性接头与N末端连接的β-2微球蛋白。这样接头的例子包括SEQ ID NO:43-166、244、307、320、355-379和383-398提供并在以下描述的接头。可溶性HLA-G1的制备见美国专利申请公开号US2004/0044182所述。
在其它实施方式中，所述HLA-G激动剂结合域是与ILT2、ILT4或KIR2DL4特异性结合的免疫球蛋白可变结合域或其衍生物(例如，抗体、Fab、scFv或类似衍生物)。具有ILT2、ILT4或KIR2DL4特异性的抗体包括例如，美国专利申请公开号US 2003/0232051中描述的抗体。
人白细胞抗原G(HLA-G)是非经典主要组织相容性复合体(MHC)I型分子，其为由重链和轻链(β-2微球蛋白)组成的异型二聚体，其中重链锚着在膜上。HLA-G作为保护胎儿组织免受母体免疫系统攻击的免疫调节分子发挥作用。尽管HLA-G组成性表达仅限于胎儿组织、成人胸腺髓质、角膜、胰岛、红细胞和内皮细胞前体，但可在癌症、移植、多发性硬化、炎性疾病和病毒感染中诱导其表达。HLA-G初级转录物产生编码膜结合蛋白同种型HLA-G1、-G2、-G3和-G4以及可溶性蛋白质同种型HLA-G5、HLA-G6和HLA-G7的7种选择性mRNA，其中HLA-G5是细胞表面结合HLA-G1蛋白的可溶性形式。
尽管HLA-G似乎不具有显著的免疫刺激功能，但已显示其结合抑制性受体，即ILT2、ILT4、KIR2DL4和CD8，并由此与B细胞、T细胞、NK细胞和抗原呈递细胞发生相互作用。HLA-G的二聚体形式对ILT2的亲和力比对ILT4、KIR2DL4或CD8的亲和力高出若干数量级。已显示HLA-G1抑制子宫和外周血NK细胞的溶细胞功能、细胞毒性T淋巴细胞的抗原特异性溶细胞功能、CD4+T细胞的同增殖(alloproliferative)应答、T细胞和外周血NK细胞的增殖、树突细胞的成熟和功能(参见例如，Wiendl等(2003)Blood,126:176-185)。已表明HLA-G可用于降低CNS中与多发性硬化相关的炎症应答(Wiendl等(2005)Blood,128:2689-2704)，并作为促进移植中移植物耐受性的治疗剂(Carosella等(2008)Blood 111:4862-4870)。
在一些实施方式中，本发明的结合域包含如本文所述具有ILT2、ILT4或KIR2DL4特异性的VL和VH结构域。在某些实施方式中，所述VL和VH结构域是人的结构域。VL和VH结构域可以任何取向排列，并可由本文所述的至多约30个氨基酸的接头或能够提供与两个亚结合域相互作用相容的间隔物功能的任何其它氨基酸序列分隔开。在某些实施方式中，连接VL和VH结构域的接头包含SEQ ID NO：43-166、244、307、320、355-379和383-398中一个或多个所示的氨基酸序列，如接头115(SEQ ID NO:148)、SEQ ID NO:244中提供的接头、接头46(SEQ IDNO:88)、接头130(SEQ ID NO:163)或接头131(SEQ ID NO:164)。多特异性结合域可具有至少2个特异性亚结合域，其类似于驼类抗体组成；或具有至少4个特异性亚结合域，其类似于成对VL和VH链的更常规哺乳动物抗体组成。
在其它实施方式中，本发明的ILT2、ILT4或KIR2DL4特异性结合域可包含一个或多个互补决定区("CDR")，或多个拷贝的一种或多种这类CDR，这类CDR是抗ILT2、抗ILT4或抗KIR2DL4的scFv或Fab片段可变区或其重链或轻链可变区获得、衍生或设计出。因此，本发明的结合域可包含来自抗ILT2、抗ILT4或抗KIR2DL4的可变区的单个CDR，或可包含多个相同或不同的CDR。
HGF
如上所述，在某些实施方式中，本发明提供含有作为HGF激动剂(即可提高HGF信号传导)的结合区或结合域的多肽。在一些实施方式中，所述HGF激动剂结合域是HGF或其功能性亚结构域。
肝细胞生长因子(HGF)通过在结合原癌基因c-Met受体后激活酪氨酸激酶信号级联放大来调节细胞生长、细胞运动性和形态发生。HGF影响多种细胞类型并调节各种生物活性，包括细胞因子的产生、细胞迁移、增殖和存活。HGF以单个无活性多肽的形式分泌，被丝氨酸蛋白酶切割为69kDa的α链和34kDa的β链。α和β链之间的二硫键结合产生活性异型二聚体分子。HGF基因的可变剪接产生5种不同的同种型(同种型1-5；分别为Genebank登录号NP_000592.3、NP_001010931.1、NP_001010932.1、NP_001010933.1和NP_001010934.1；SEQ IDNO:18-22；这些序列中各自的氨基酸1-31是信号肽)。
HGF被认为是预防和弱化疾病发展的关键因子(Ito等(2008)Int.Arch.AllergyImmunol.146Suppl 1:82-87)。例如，已显示HGF在小鼠中有效抑制胶原诱导的关节炎(Okunishi等(2007)Jnl.Immunol.179:5504-5513)，在变应性气道炎症的小鼠模型中发挥保护作用(Okunishi等(2005)Jnl.Immunol.175:4745-4753；Ito等Am.J.Respir.Cell.Mol.Biol.(2005)32:268-280)。
在一些实施方式中，本发明的结合域包含如本文所述具有HGF特异性的VL和VH结构域。在某些实施方式中，所述VL和VH结构域是人的结构域。VL和VH结构域可以任何取向排列，并可由本文所述的至多约30个氨基酸的接头或能够提供与两个亚结合域相互作用相容的间隔物功能的任何其它氨基酸序列分隔开。在某些实施方式中，连接VL和VH结构域的接头包含SEQ ID NO：43-166、244、307、320、355-379和383-398中一个或多个所示的氨基酸序列，如SEQ ID NO:244中提供的接头、接头46(SEQ ID NO:88)、接头130(SEQ ID NO:163)或接头131(SEQ ID NO:164)。多特异性结合域可具有至少2个特异性亚结合域，其类似于驼类抗体组成；或具有至少4个特异性亚结合域，其类似于成对VL和VH链的更常规哺乳动物抗体组成。在其它实施方式中，本发明的HGF特异性结合域可包含一个或多个互补决定区("CDR")，或者多个拷贝的一种或多种这类CDR，这类CDR由抗HGF的scFv或Fab片段可变区或其重链或轻链可变区获得、衍生或设计出。因此，本发明的结合域可包含来自抗HGF可变区的单个CDR，或其可包含多个相同或不同的CDR。在某些实施方式中，本发明的结合域包含具有HGF特异性的VL和VH结构域，其包含框架区以及CDR1、CDR2和CDR3区。
IL35
如上所述，在某些实施方式中，本发明提供含有作为IL35激动剂(即可提高IL35信号传导)的结合区或结合域的多肽。在某些实施方式中，所述IL35激动剂结合域是IL35(例如，SEQ ID NO:25和26)或其功能性亚结构域。在某些实施方式中，所述IL35激动剂结合域是包含SEQ ID NO:25和26序列的单链多肽或其亚结构域。这样的单链多肽可包含一个或多个接头，包括本文所述的接头。在其它实施方式中，所述IL35激动剂结合域是具有IL35激动剂活性的IL35R特异性的单链免疫球蛋白可变结构域，如scFv。
IL-35是IL-12细胞因子亚家族的新近描述的细胞因子。异型二聚体分子由IL-12p35和IL-27Ebi3亚基组成。近期已显示其在关节炎小鼠模型中为Treg功能的强效诱导物，且能够改变TH17应答(Niedbala等(2007)Eur.J.Immunol.37:3021；Collison等(2007)Nature 450:566)。因此，预测IL-35激动作用和CD86抑制作用的结合可增加单独的CD28抑制作用的治疗益处。
调节T细胞(TREGS)是CD4+T细胞的关键亚群，其对保持自身耐受和预防自身免疫、限制慢性炎性疾病如哮喘和炎性肠病、调节稳态淋巴细胞扩增具有重要作用。IL35是抗炎细胞因子，已显示其通过刺激调节T细胞扩增和抑制Th17细胞发育来抑制免疫应答(Collison等(2007)Nature450:566-9)。IL35是由EB病毒诱导基因3(EBI3；SEQ ID NO:25；信号肽：氨基酸1-20)和IL12的p35亚基(SEQ ID NO:26；信号肽：氨基酸1-56)形成的异型二聚体(Devergne等(1997)Proc.Natl.Acad.Sci.USA94:12041-12046；美国专利5,830,451；美国专利申请公开号US2007/0299026)。已显示其在鼠胶原诱导关节炎模型中具有与EnbrelTM等效的治疗作用(Niedbala等(2007)Eur.J.Immunol.37:3021-3029)，因此已提出将其作为对临床类风湿性关节炎的治疗剂。
在一些实施方式中，本发明的结合域包含如本文所述具有IL35R特异性的VL和VH结构域。在某些实施方式中，所述VL和VH结构域是人的结构域。VL和VH结构域可以任何取向排列，并可由本文所述的至多约30个氨基酸的接头或能够提供与两个亚结合域相互作用相容的间隔物功能的任何其它氨基酸序列分隔开。在某些实施方式中，连接VL和VH结构域的接头包含SEQ ID NO：43-166、244、307、320、355-379和383-398中一个或多个所示的氨基酸序列，如SEQ ID NO:244中提供的接头、接头46(SEQ ID NO:88)、接头130(SEQ ID NO:163)或接头131(SEQ ID NO:164)。多特异性结合域可具有至少2个特异性亚结合域，其类似于驼类抗体组成；或具有至少4个特异性亚结合域，其类似于成对VL和VH链的更常规哺乳动物抗体组成。在其它实施方式中，本发明的IL35R特异性结合域可包含一个或多个互补决定区("CDR")，或者多个拷贝的一种或多种这类CDR，这类CDR由抗IL35R的scFv或Fab片段可变区或其重链或轻链可变区获得、衍生或设计出。因此，本发明的结合域可包含来自抗IL35R可变区的单个CDR，或其可包含多个相同或不同的CDR。在某些实施方式中，本发明的结合域包含具有IL-35R特异性的VL和VH结构域，其包含框架区以及CDR1、CDR2和CDR3区。
LIGHT
如上所述，在某些实施方式中，本发明提供含有作为LIGHT拮抗物(即可抑制LIGHT信号传导)的结合区或结合域的多肽。在一些实施方式中，所述LIGHT拮抗物结合域是HVEM胞外域(也称为sHVEM；SEQ ID NO:29；信号肽：氨基酸1-38)或其功能性亚结构域。在其它实施方式中，所述LIGHT拮抗物结合域是LIGHT特异性单链免疫球蛋白样可变结构域，如scFv。在某些实施方式中，所述LIGHT拮抗物结构域是包含PCT专利申请公开号WO 08/027338所述VH和VL结构域的单链免疫球蛋白样可变结构域。
LIGHT是表达于活化T淋巴细胞、单核细胞、粒细胞和未成熟树突细胞的TNF超家族成员。已报道了LIGHT的两种不同的同种型(Genebank登录号NP_003798.2和NP_742011.1)。已显示LIGHT通过疱疹病毒进入介导物(HVEM)和淋巴毒素β受体(LTβR)信号传导调节T细胞免疫应答。HVEM和LTβR均与LIGHT发生高亲和力结合，其中在T细胞、B细胞、天然杀伤细胞和内皮细胞检测到HVEM表达，LTβR在单核细胞和基质细胞但非T细胞和B细胞中表达。已显示LIGHT是CD28依赖性T细胞激活作用的共刺激分子，其优先诱导IFN-γ和GM-CSF的产生。在鼠模型中通过体内给予LTβR-Ig融合蛋白或抗LIGHT抗体阻断LIGHT导致T细胞介导的免疫应答降低，改善移植物抗宿主疾病(Tamada等(2000)Nat.Med.6:283-9)。LIGHT的组成性表达导致组织破坏和自身免疫样疾病综合征(Granger和Rickert(2003)Cytokine GrowthFactor Rev.14:289-96)。
在一些实施方式中，本发明的结合域包含如本文所述具有LIGHT特异性的VL和VH结构域。在某些实施方式中，所述VL和VH结构域是人的结构域。VL和VH结构域可以任何取向排列，并可由本文所述的至多约30个氨基酸的接头或能够提供与两个亚结合域相互作用相容的间隔物功能的任何其它氨基酸序列分隔开。在某些实施方式中，连接VL和VH结构域的接头包含SEQ ID NO：43-166、244、307、320、355-379和383-398中一个或多个所示的氨基酸序列，如SEQ ID NO:244中提供的接头、接头46(SEQ ID NO:88)、接头130(SEQ ID NO:163)或接头131(SEQ ID NO:164)。多特异性结合域可具有至少2个特异性亚结合域，其类似于驼类抗体组成；或具有至少4个特异性亚结合域，其类似于成对VL和VH链的更常规哺乳动物抗体组成。
在其它实施方式中，本发明的LIGHT特异性结合域可包含一个或多个互补决定区("CDR")，或者多个拷贝的一种或多种这类CDR，这类CDR由抗LIGHT的scFv或Fab片段可变区或其重链或轻链可变区获得、衍生或设计出。因此，本发明的结合域可包含来自抗LIGHT可变区的单个CDR，或其可包含多个相同或不同的CDR。在某些实施方式中，本发明的结合域包含LIGHT特异性VL和VH结构域，其包含框架区以及CDR1、CDR2和CDR3区。
PD-1
如上所述，在某些实施方式中，本发明提供含有作为PD-1激动剂(即可提高PD-1信号转导)的结合区或结合域的多肽。在一些实施方式中，所述PD-1激动剂结合域是PD1-L1(例如，SEQ ID NO:32；信号肽：氨基酸1-18)、PD1-L2(例如，SEQ ID NO:33；信号肽：氨基酸1-19)或其功能性亚结构域。在其它实施方式中，所述PD-1激动剂结合域是PD-1特异性单链免疫球蛋白样可变结构域，如scFv。PD-1特异性抗体包括例如，美国专利申请公开号US2006/0210567所述的抗体。
PD-1(Genebank登录号NP_005009.1)是表达于活化T细胞、B细胞和髓样细胞的CD28/CTLA4家族成员。PD-1包含免疫受体基于酪氨酸的抑制基序。PD-1通过与程序性死亡1配体1(PD1-L1；也称为CD274)和程序性死亡1配体2(PD1-L2)结合发挥作用。人的PD-L1和PD-L2同时表达于未成熟和成熟的树突细胞、IFN-γ处理的单核细胞和滤泡性树突细胞上。PD-1缺陷小鼠显示各种自身免疫病理学，表明PD-1是免疫应答的负调节物(Nishimura和Honjo(2001)Trends Immunol.2:265；Nishimura等(1999)Immunity 11:141)。已显示PD-1与PD1-L1和PD1-L2的结合引起T细胞激活作用下调(Freeman等(2000)J.Exp.Med.192:1027；Latchman等(2001)Nat.Immunol.2:261；Carter等(2002)Eur.J.Immunol.32:634)。
在一些实施方式中，本发明的结合域包含如本文所述具有PD-1特异性的VL和VH结构域。在某些实施方式中，所述VL和VH结构域是人的结构域。VL和VH结构域可以任何取向排列，并可由本文所述的至多约30个氨基酸的接头或能够提供与两个亚结合域相互作用相容的间隔物功能的任何其它氨基酸序列分隔开。在某些实施方式中，连接VL和VH结构域的接头包含SEQ ID NO：43-166、244、307、320、355-379和383-398中所示的氨基酸序列，如SEQ IDNO:244中提供的接头、接头46(SEQ ID NO:88)、接头130(SEQ ID NO:163)或接头131(SEQID NO:164)。多特异性结合域可具有至少2个特异性亚结合域，其类似于驼类抗体组成；或具有至少4个特异性亚结合域，其类似于成对VL和VH链的更常规哺乳动物抗体组成。
在其它实施方式中，本发明的PD-1特异性结合域可包含一个或多个互补决定区("CDR")，或者多个拷贝的一种或多种这类CDR，这类CDR由抗PD-1的scFv或Fab片段可变区或其重链或轻链可变区获得、衍生或设计出。因此，本发明的结合域可包含来自抗PD-1可变区的单个CDR，或其可包含多个相同或不同的CDR。
BTLA
如上所述，在某些实施方式中，本发明提供含有作为BTLA激动剂(即可提高BTLA信号传导)的结合区或结合域的多肽。在一些实施方式中，所述BTLA激动剂结合域是HVEM胞外域(也称为sHVEM；SEQ ID NO:29；信号肽：氨基酸1-38)或其功能性亚结构域(例如SEQ IDNO:29的氨基酸54-78)。在其它实施方式中，所述BTLA激动剂结合域是BTLA特异性单链免疫球蛋白样可变结构域，如scFv。BTLA特异性激动剂抗体见例如Krieg等(2005)J.Immunol.175:6420-6472所述。
BTLA(Genebank登录号NP_001078826.1和NP_861445.3；分别为同种型2和1)是作为免疫球蛋白家族成员的细胞表面蛋白，表达于B细胞、T细胞和抗炎呈递细胞。BTLA的配体是疱疹病毒进入介导物(HVEM)，其为肿瘤坏死因子受体家族的成员并作为LIGHT的配体(Sedy等(2005)Nat.Immunol.6:90-98)。已在HVEM的CRD1中鉴定出BTLA结合位点(SEQ IDNO:29的氨基酸54-78；PCT专利申请公开号WO 2006/063067).该位点不同于LIGHT所用的位点但与HVEM的gD结合位点重叠。尽管HVEM与LIGHT结合诱导强免疫应答，但HVEM与BTLA结合产生对T细胞应答的负调节作用(Murphy等(2006)Nat.Rev.Immunol.6:671-681)。已显示BTLA与HVEM结合激活BTLA的酪氨酸磷酸化，由此诱导与蛋白质酪氨酸磷酸酶SHP-1和SHP-2结合(Gavrieli等(2003)Biochem.Biophys.Res.Commun.312:1236)，但一些数据对SHP募集是否负责BTLA的负调节活性存在疑问(Chemnitz等(2006)J.Immunol.176:6603-6614)。
已显示可溶性HVEM抑制抗CD3诱导的CD4+T细胞增殖，该作用被抗BTLA抗体逆转(Gonzalez等(2005)Proc.Natl.Acad.Sci.USA102:1116-1121)。类似地，显示激动性抗BTLA单克隆抗体抑制抗CD3介导的CD4+T细胞增殖和细胞因子产生(Krieg等(2005)J.Immunol.175:6420-6472)。缺乏完整BTLA基因的小鼠显示对实验性自身免疫脑脊髓炎的敏感性升高(Watanabe等(2003)Nat.Immunol.4:670-679)和气道炎症延长(Deppong等(2006)J.Immunol.176:3909-3913)。
在一些实施方式中，本发明的结合域包含如本文所述具有BTLA特异性的VL和VH结构域。在某些实施方式中，所述VL和VH结构域是人的结构域。VL和VH结构域可以任何取向排列，并可由本文所述的至多约30个氨基酸的接头或能够提供与两个亚结合域相互作用相容的间隔物功能的任何其它氨基酸序列分隔开。在某些实施方式中，连接VL和VH结构域的接头包含SEQ ID NO：43-166、244、307、320、355-379和383-398中所示的氨基酸序列，如SEQ IDNO:244中提供的接头、接头46(SEQ ID NO:88)、接头130(SEQ ID NO:163)或接头131(SEQID NO:164)。多特异性结合域可具有至少2个特异性亚结合域，其类似于驼类抗体组成；或具有至少4个特异性亚结合域，其类似于成对VL和VH链的更常规哺乳动物抗体组成。
在其它实施方式中，本发明的BTLA特异性结合域可包含一个或多个互补决定区("CDR")，或者多个拷贝的一种或多种这类CDR，这类CDR由抗BTLA的scFv或Fab片段可变区或其重链或轻链可变区获得、衍生或设计出。因此，本发明的结合域可包含来自抗BTLA可变区的单个CDR，或其可包含多个相同或不同的CDR。
GITRL
如上所述，在某些实施方式中，本发明提供含有作为GITRL拮抗物(即可抑制GITRL信号传导)的结合区或结合域的多肽。在一些实施方式中，所述GITRL拮抗物结合域是GITR胞外域(也称为sGITR；SEQ ID NO:39和40；这些序列各自的信号肽：氨基酸1-25)或其功能性亚结构域。在其它实施方式中，所述GITRL拮抗物结合域是GITRL特异性单链免疫球蛋白样可变结构域，如scFv。GITRL的拮抗物抗体见例如美国专利申请公开号2005/0014224所述。
糖皮质激素诱导的肿瘤坏死因子受体(GITR；也称为AITR)是I型跨膜蛋白和TNF受体超家族的成员(Nocentini等，(2007)Eur.J.Immunol.37:1165-69)。GITR在T细胞增殖和TCR介导的凋亡中发挥重要作用。GITR表达在T细胞上上调，高水平的GITR在CD4+CD25+调节T细胞上组成性表达(Kwon等(2003)Exp.Mol.Med.35:8-16)，表达也发生在巨噬细胞、B细胞和NK细胞上(Liu等(2008)J.Biol.Chem.283:8202-8210)。GITR的关联配体GITRL在抗原呈递细胞如树突细胞和B细胞上组成性表达。已显示GITR与GITRL的结合使CD4+CD25-效应物T细胞对CD4+CD25+调节T细胞的抑制作用具有耐受性。已显示通过GITRL或激动剂抗体激活GITR增加TCR诱导的T细胞增殖和细胞因子产生，并拯救T细胞免于发生抗CD3诱导的凋亡(Nocentini et al.(1997)Proc.Natl.Acad.Sci.USA94:6216-6221)。此外，GITR与GITRL结合可抑制T调节细胞和/或使效应物T细胞对T调节细胞介导的抑制作用的耐受性增大(Kanamaru等(2004)J.Immunol.172:7306-7314)。
研究表明，在实验性自身免疫脑脊髓炎的诱导期给予抗GITR显著增强临床疾病的严重程度并增加CNS炎症和自身反应T细胞应答(Kohm et al.(2004)J.Immunol.172:4686-4690)。此外，激活GITR信号传导加剧鼠哮喘和胶原诱导的关节炎(Patel等(2005)Eur.J.,Immunol.35:3581-90)。
在一些实施方式中，本发明的结合域包含如本文所述具有GITRL特异性的VL和VH结构域。在某些实施方式中，所述VL和VH结构域是人的结构域。VL和VH结构域可以任何取向排列，并可由本文所述的至多约30个氨基酸的接头或能够提供与两个亚结合域相互作用相容的间隔物功能的任何其它氨基酸序列分隔开。在某些实施方式中，连接VL和VH结构域的接头包含SEQ ID NO：43-166、244、307、320、355-379和383-398中所示的氨基酸序列，如SEQ IDNO:244中提供的接头、接头46(SEQ ID NO:88)、接头130(SEQ ID NO:163)或接头131(SEQID NO:164)。多特异性结合域可具有至少2个特异性亚结合域，其类似于驼类抗体组成；或具有至少4个特异性亚结合域，其类似于成对VL和VH链的更常规哺乳动物抗体组成。
在其它实施方式中，本发明的GITRL特异性结合域可包含一个或多个互补决定区("CDR")，或者多个拷贝的一种或多种这类CDR，这类CDR由抗GITRL的scFv或Fab片段可变区或其重链或轻链可变区获得、衍生或设计出。因此，本发明的结合域可包含来自抗GITRL可变区的单个CDR，或其可包含多个相同或不同的CDR。在某些实施方式中，本发明的结合域包含GITRL特异性VL和VH结构域，其包含框架区以及CDR1、CDR2和CDR3区。
CD40
如上所述，在某些实施方式中，本发明提供含有作为CD40拮抗物(即可抑制CD40信号传导)的结合区或结合域的多肽。在一些实施方式中，所述CD40拮抗物结合域是CD40特异性单链免疫球蛋白样可变结构域，如scFv。CD40的拮抗物抗体见例如美国专利申请公开号US 2008/0057070和美国专利5,874,082和6,838,261所述。
CD40是发现于正常和肿瘤性B细胞、树突细胞、抗炎呈递细胞、内皮细胞、单核细胞和上皮细胞表面上的55kDa的细胞表面抗原。CD40在抗原呈递细胞上的表达在辅助性T细胞和细胞毒性T淋巴细胞的作用中发挥重要的共刺激作用。CD40配体(CD40L，也称为CD154)在T细胞上的表达在正常免疫应答过程中上调。表达于T细胞的CD40L与表达于B细胞的CD40的结合引起B细胞增殖和分化、抗体产生、同种型转换和B细胞记忆产生。已显示人的抗CD40拮抗性抗体对人慢性淋巴细胞性白血病细胞具有抗白血病活性(Luqman等(2008)Blood 112:711-720)。
在一些实施方式中，本发明的结合域包含如例如美国专利申请公开号US 2008/0057070所述具有CD40特异性的VL和VH结构域。在某些实施方式中，所述VL和VH结构域是人的结构域。VL和VH结构域可以任何取向排列，并可由本文所述的至多约30个氨基酸的接头或能够提供与两个亚结合域相互作用相容的间隔物功能的任何其它氨基酸序列分隔开。在某些实施方式中，连接VL和VH结构域的接头包含SEQ ID NO：43-166、244、307、320、355-379和383-398中所示的氨基酸序列，如SEQ ID NO:244中提供的接头、接头46(SEQ ID NO:88)、接头130(SEQ ID NO:163)或接头131(SEQ ID NO:164)。多特异性结合域可具有至少2个特异性亚结合域，其类似于驼类抗体组成；或具有至少4个特异性亚结合域，其类似于成对VL和VH链的更常规哺乳动物抗体组成。
在其它实施方式中，本发明的CD40特异性结合域可包含一个或多个互补决定区("CDR")，或者多个拷贝的一种或多种这类CDR，这类CDR由抗CD40的scFv或Fab片段可变区或其重链或轻链可变区获得、衍生或设计出。因此，本发明的结合域可包含来自抗CD40可变区的单个CDR，或其可包含多个相同或不同的CDR。在一些实施方式中，本发明的结合域包含如例如美国专利申请公开号US 2008/0057070所述具有CD40特异性的VL和VH结构域，其包含框架区以及CDR1、CDR2和CDR3区。
多特异性融合蛋白
本发明提供包含结合CD86的结构域(“CD86结合域”)和结合非CD86分子的结构域(“异源结合域”)的多特异性融合蛋白。在某些实施方式中，所述异源结合域是IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物。
在某些实施方式中，所述异源结合域是IL-10激动剂，如IL10、IL10Fc或与IL10R1或IL10R2特异性结合的单链结合域。在某些实施方式中，所述异源结合域是HLA-G激动剂，如HLA-G1、HLA-G5、HLA-G突变蛋白或其功能区(如胞外域)、或与ILT2、ILT4或KIR2DL4特异性结合的单链结合域。在某些实施方式中，所述异源结合域是HGF激动剂，如HGF或其亚结构域。在某些实施方式中，所述异源结合域是IL-35激动剂，如IL35或其亚结构域、单链IL-35或其亚结构域、或具有IL35R特异性和IL35激动剂活性的单链免疫球蛋白样可变结构域。在某些实施方式中，所述异源结合域是LIGHT拮抗物，如HVEM胞外域或其亚结构域、或具有LIGHT特异性的单链免疫球蛋白样可变结构域。在某些实施方式中，所述异源结合域是PD-1激动剂，如PD1-L1、PD1-L2或其亚结构域、或具有PD-1特异性的单链免疫球蛋白样可变结构域。在某些实施方式中，所述异源结合域是BTLA激动剂，如HVEM胞外域或其亚结构域、或具有BTLA特异性的单链免疫球蛋白样可变结构域。在某些实施方式中，所述异源结合域是GITRL拮抗物，如GITR胞外域或其亚结构域、或具有GITRL特异性的单链免疫球蛋白样可变结构域。在某些实施方式中，所述异源结合域是CD40拮抗物，如具有CD40特异性的单链免疫球蛋白样可变结构域。
通常，本发明的融合蛋白利用不含前导肽(信号肽)的成熟蛋白质。因此，尽管本文提供的某些结合域蛋白序列(如本文所述的CTLA4、CD28、HLA-G1、HLA-G5和其它)包括前导肽，技术人员很容易理解如何从包含信号肽的序列确定成熟蛋白质序列。在某些实施方式中，包含前导肽可能具有实用性。
考虑到CD86结合域可位于融合蛋白的氨基末端，异源结合域可位于羧基末端。在某些实施方式中，交叉受体分子如SEQ ID NO:9、13、17、24、28、31、35、42、171、173、175、177、179、181、187、189、191、193、219、221、223、237、262、302、330、336、338、340或400所示。还考虑到异源结合域可位于氨基末端，而CD86结合域可位于羧基末端。在某些实施方式中，交叉受体分子如SEQ ID NO:183、185、199、201、203、205、207、211、213、254、258、266、276、350、352或354所示。如本文所述，本发明的结合域可与间插结构域(例如免疫球蛋白恒定区或其亚区)的任一端融合。此外，两个或多个结合域可各自通过本文所述接头与间插结构域连接。
本文所用的“间插结构域”指一种氨基酸序列，其简单地用作一个或多个结合域的支架以使融合蛋白主要(例如，50％或更多融合蛋白)或基本(例如，90％或更多融合蛋白)以单链多肽形式存在于组合物中。例如，某些间插结构域可具有结构功能(例如间隔、柔性、刚性)或生物学功能(例如在血浆，如人血中半衰期延长)。可延长本发明融合蛋白的血浆半衰期的示例性间插结构域包括清蛋白、转铁蛋白、结合血清蛋白的支架结构域等，或其片段。
在某些实施方式中，本发明多特异性融合蛋白所含的间插结构域是“二聚化结构域”，其指能够通过非共价或共价相互作用，例如通过形成氢键、静电相互作用、范德华力、二硫键、疏水相互作用等或其任何组合促进至少两种单链多肽或蛋白质结合的氨基酸序列。示例性二聚化结构域包括免疫球蛋白重链恒定区或亚区。应理解，二聚化结构域可促进二聚体或更高级多聚体复合物(如三聚体、四聚体、五聚体、六聚体、七聚体、八聚体等)的形成。
本文定义的术语“恒定亚区”指对应于或衍生自来源抗体一个或多个恒定区结构域的部分或全部，但并非所有恒定区结构域的肽、多肽或蛋白质序列。在一个优选实施方式中，所述恒定亚区是IgG CH2CH3，优选IgG1CH2CH3。在一些实施方式中，本发明的融合蛋白恒定区结构域可能没有或具有很少的抗体依赖性细胞介导的细胞毒性(ADCC)、补体激活和补体依赖性细胞毒性(CDC)的效应物功能，同时保持结合一些FC受体(如FCRn结合)的能力并保持较长的体内半衰期。在某些实施方式中，本发明的结合域与人IgG1恒定区或亚区融合，其中所述IgG1恒定区或亚区中具有一个或多个以下突变氨基酸：第234位的亮氨酸(L234)、第235位的亮氨酸(L235)、第237位的甘氨酸(G237)、第318位的谷氨酸(E318)、第320位的赖氨酸(K230)、第322位的赖氨酸(K322)或其任何组合(根据Kabat编号)。例如，可将这些氨基酸中的任何一个或多个改变为丙氨酸。在另一个实施方式中，IgG1Fc结构域中L234、L235、G237、E318、K320和K322(根据EU编号)各突变为丙氨酸(即分别为L234A、L235A、G237A、E318A、K320A和K322A)，并任选具有N297A突变(即基本消除CH2结构域的糖基化)。
本领域已知在Fc结构域内部或外部进行可改变Fc与Fc受体(CD16、CD32、CD64、CD89、FcεR1、FcRn)或补体组分C1q相互作用的突变的方法(参见例如，美国专利号5,624,821；Presta(2002)Curr.Pharma.Biotechnol.3:237)。本发明的具体实施方式包括含有具有来自人IgG的恒定区或亚区的免疫球蛋白或融合蛋白的组合物，其中与FcRn和蛋白A的结合被保留，且Fc结构域不再与其它Fc受体或C1q发生相互作用或与最大程度减少相互作用。例如，本发明结合域可与人IgG1恒定区或亚区融合，其中第297位的天冬酰胺(根据Kabat编号为N297)突变为另一种氨基酸，以减少或消除该位点的糖基化，因此消除了Fc与FcγR和C1q的有效结合。另一个示例性突变是P331S，其消除了C1q结合但不影响Fc结合。
在其它实施方式中，免疫球蛋白Fc区可能相对于免疫球蛋白参比序列具有改变的糖基化模式。例如，可采用各种遗传技术来改变构成糖基化位点的一个或多个特定氨基酸残基(参见Co等(1993)Mol.Immunol.30:1361；Jacquemon等(2006)J.Thromb.Haemost.4:1047；Schuster等(2005)Cancer Res.65:7934；Warnock等(2005)Biotechnol.Bioeng.92:831)，如CH2结构域的N297(EU编号)。或者，可工程改造产生本发明融合蛋白的宿主细胞，以产生改变的糖基化模式。例如，本领域已知的一种方法提供改变的糖基化模式，采用平分的非岩藻糖基化变体形式提高ADCC。所述变体由含有寡糖修饰酶的宿主细胞表达产生。或者，考虑用BioWa/Kyowa Hakko的Potelligent技术降低本发明糖基化分子的岩藻糖含量。在一种已知方法中，提供了用于产生重组免疫球蛋白的CHO宿主细胞，其通过产生GDP-岩藻糖改变免疫球蛋白Fc区的糖基化模式。
或者，采用化学技术改变本发明融合蛋白的糖基化模式。例如，各种糖苷酶和/或甘露糖苷酶抑制剂提供增加ADCC活性、提高Fc受体结合和改变糖基化模式中的一种或多种所需效果。在某些实施方式中，在含有浓度能提高所述宿主细胞产生的免疫糖蛋白分子的ADCC的碳水化合物调节物的培养基中培养表达本发明多特异性融合蛋白(含有与IL10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物连接的CD86拮抗物)的细胞，其中所述碳水化合物调节物的浓度小于800μM。在一个优选实施方式中，在含有浓度为100-800μM，如100μM、200μM、300μM、400μM、500μM、600μM、700μM或800μM的澳粟精胺或几夫碱，更优选澳粟精胺的培养基中培养表达这些多特异性融合蛋白的细胞。美国专利申请公开号2009/0041756或PCT公开号WO 2008/052030中提供了用碳水化合物调节物如澳粟精胺改变糖基化的方法。
在另一个实施方式中，免疫球蛋白Fc区可具有影响与效应物细胞Fc受体结合的氨基酸修饰。可采用本领域已知的任何技术，如Presta等(2001)Biochem.Soc.Trans.30:487所公开的方法进行这些修饰。在另一种方法中，可使用Xencor XmAbTM技术工程改造对应于Fc结构域的恒定亚区，以增强细胞杀伤性效应物功能(参见Lazar等(2006)Proc.Nat'l.Acad.Sci.(USA)103:4005)。例如，可使用这种方法产生对FCγR的特异性和结合改进的恒定亚区，从而增强细胞杀伤性效应物功能。
在其它实施方式中，与缺少这类间插结构域的相应融合蛋白相比，恒定区或亚区可任选地增加血浆半衰期或胎盘转移。在某些实施方式中，本发明融合蛋白在人体内的延长的血浆半衰期是至少2、至少3、至少4、至少5、至少10、至少12、至少18、至少20、至少24、至少30、至少36、至少40、至少48小时，至少数日，至少一周，至少2周，至少数周，至少一个月，至少两个月，至少数月，或更长时间。
恒定亚区可包含任何以下结构域的部分或全部：CH2结构域、CH3结构域(IgA、IgD、IgG、IgE或IgM)和CH4结构域(IgE或IgM)。因此，本文定义的恒定亚区可指对应于免疫球蛋白恒定区一部分的多肽。恒定亚区可包括衍生自相同或不同的免疫球蛋白、抗体同种型或等位基因变体的CH2结构域和CH3结构域。在一些实施方式中，CH3结构域是截短形式，其包含美国专利申请号12/041,590(PCT/US2007/071052的CIP)列出的如SEQ ID NOS:366-371所示的的羧基末端序列。在某些实施方式中，本发明多肽的恒定亚区具有CH2结构域和CH3结构域，其可任选具有氨基末端接头、羧基末端接头或在两端均具有接头。
“接头”是结合或连接其它肽或多肽的肽，如约2至150个氨基酸的接头。在本发明的融合蛋白中，接头可将间插结构域(例如免疫球蛋白衍生的恒定亚区)与结合域连接，或接头可连接结合域的两个可变区或从异型二聚体分子形成的单链多肽内的两个区域，如EBI3(SEQ ID NO:25)和IL12(SEQ ID NO:26)或IL35的p35亚基。例如，接头可以是由抗体绞链区序列获得、产生或设计出的氨基酸序列，将结合域与受体连接的序列，或是将结合域与细胞表面跨膜区或膜锚定物连接的序列。在一些实施方式中，接头可具有至少一个能够在生理条件或其它标准肽条件(例如，肽纯化条件、肽储存条件)下参与至少一个二硫键的半胱氨酸。在某些实施方式中，对应于或类似于免疫球蛋白铰链肽的接头保留了对应于置于朝向该铰链氨基末端的铰链半胱氨酸的半胱氨酸。在其它实施方式中，接头来自IgG1或IgG2A铰链，并含有对应于铰链半胱氨酸的一个半胱氨酸或两个半胱氨酸。在某些实施方式中，在间插结构域之间形成一个或多个二硫键，作为链间二硫键。在其它实施方式中，本发明的融合蛋白可具有直接与结合域融合的间插结构域(即不存在接头或铰链)。在一些实施方式中，所述间插结构域是二聚化结构域，如IgG1CH2CH3Fc部分。
本发明多特异性融合蛋白的间插或二聚化结构域可通过肽接头与一个或多个末端结合域连接。除提供间隔功能以外，接头可在融合蛋白内以及融合蛋白和其靶点之间提供适合将融合蛋白的一个或多个结合域适当定向的柔性或刚性。此外，在给予有需要的对象如人后，接头可在体外和体内支持全长融合蛋白的表达和纯化蛋白的稳定性，且接头在这些相同对象中优选无免疫原性或免疫原性较低。在某些实施方式中，本发明多特异性融合蛋白的间插或二聚化结构域的接头可包含人免疫球蛋白铰链的部分或全部。
此外，结合域可包含VH和VL结构域，这些可变区结构域可通过接头结合。示例性可变区结合域接头包括属于(GlynSer)家族的接头，如(Gly3Ser)n(Gly4Ser)1、(Gly3Ser)1(Gly4Ser)n、(Gly3Ser)n(Gly4Ser)n或(Gly4Ser)n，其中n是1-5的整数(参见例如，分别对应于SEQ ID NO：64、71、88、131、132、149、163和164的接头22、29、46、89、90、116、130和131)。在优选实施方式中，这些基于(Gly4Ser)的接头用于连接可变结构域，不用于将结合域(例如scFv)与间插结构域(例如IgG CH2CH3)连接。
可用于将间插结构域(例如，免疫球蛋白衍生的恒定亚区)与结合域连接的示例性接头或可连接结合域的两个可变区的接头见SEQ ID NO：43-166、244、307、320、355-379和383-398所示。
本发明考虑的接头包括例如，衍生自免疫球蛋白超家族成员的任何域间区域(例如抗体绞链区)或II型膜蛋白家族C型凝集素的柄区域的肽。这些接头的长度可以是约2到约150个氨基酸、约2到约40个氨基酸、或约8到约20个氨基酸，优选约10到约60个氨基酸，更优选约10到约30个氨基酸，最优选约15到约25个氨基酸。例如，接头1的长度为2个氨基酸，接头116的长度为36个氨基酸(接头1-133分别见SEQ ID NO:43-166所示；其它示例性接头见SEQ ID NO:244、307、320、355-379和383-398所示)。
除通常的长度考虑外，适用于本发明融合蛋白的接头包括选自下组的抗体绞链区：IgG绞链、IgA绞链、IgD绞链、IgE绞链或其变体。在某些实施方式中，接头可以是选自人IgG1、人IgG2、人IgG3、人IgG4、其片段或变体的抗体绞链区(上部和核心区域)。本文所用的作为“免疫球蛋白绞链区”的接头指在CH1羧基端和CH2氨基端(就IgG、IgA和IgD而言)或CH3氨基端(就IgE和IgM而言)之间发现的氨基酸。本文所用的“野生型免疫球蛋白绞链区”指在抗体重链中发现的置于CH1和CH2区之间并连接这两个区域(就IgG、IgA和IgD而言)或置于CH2和CH3区之间并连接这两个区域(就IgE和IgM而言)的天然产生的氨基酸序列。在优选实施方式中，野生型免疫球蛋白绞链区序列是人的序列。
根据结晶图象研究，可按照功能和结构将IgG绞链结构域再分成以下三个区域：上部绞链区、核心或中部绞链区和下部绞链区(Shin等，(1992)Immunological Reviews 130:87)。示例性上部绞链区包括IgG1中发现的EPKSCDKTHT(SEQ ID NO:383)、IgG2中发现的ERKCCVE(SEQ ID NO:384)、IgG3中发现的ELKTPLGDTTHT(SEQ ID NO:385)或EPKSCDTPPP(SEQ ID NO:386)、以及IgG4中发现的ESKYGPP(SEQ ID NO:387)。示例性中部绞链区包括IgG1和IgG2中发现的CPPCP(SEQ ID NO:398)、IgG3中发现的CPRCP(SEQ ID NO:388)和IgG4中发现的CPSCP(SEQ ID NO:389)。尽管IgG1、IgG2和IgG4抗体各显示具有一个上部和中部铰链，IgG3具有4个串联铰链——1个ELKTPLGDTTHTCPRCP(SEQ ID NO:390)和3个EPKSCDTPPPCPRCP(SEQ ID NO:391)。
IgA和IgD抗体显示缺少IgG-样核心区，IgD显示具有两个串联的上部绞链区(参见SEQ ID NO:392和393)。发现于IgA1和IgA2抗体的示例性野生型上部绞链区分别见SEQ IDNO:394和395所示。
相反，IgE和IgM抗体包含具有铰链样性质的CH2区而非典型的绞链区。IgE和IgM的示例性野生型CH2上部铰链样序列分别见SEQ ID NO:396(VCSRDFTPPT VKILQSSSDGGGHFPPTIQL LCLVSGYTPG TINITWLEDG QVMDVDLSTA STTQEGELAS TQSELTLSQK HWLSDRTYTCQVTYQGHTFE DSTKKCA)和SEQ ID NO:397(VIAELPPKVS VFVPPRDGFF GNPRKSKLICQATGFSPRQI QVSWLREGKQ VGSGVTTDQV QAEAKESGPT TYKVTSTLTI KESDWLGQSM FTCRVDHRGLTFQQNASSMC VP)所示。
“改变的野生型免疫球蛋白绞链区”或“改变的免疫球蛋白绞链区”指(a)含有最多30％氨基酸改变(例如，最多25％、20％、15％、10％或5％氨基酸取代或缺失)的野生型免疫球蛋白绞链区，(b)长度为至少10个氨基酸(例如，至少12、13、14或15个氨基酸)、含有最多30％氨基酸改变(例如，最多25％、20％、15％、10％或5％氨基酸取代或缺失)的野生型免疫球蛋白绞链区的一部分，或(c)包含核心绞链区的野生型免疫球蛋白绞链区的一部分(该部分长度可以是4、5、6、7、8、9、10、11、12、13、14或15个氨基酸，或至少4、5、6、7、8、9、10、11、12、13、14或15个氨基酸)。在某些实施方式中，野生型免疫球蛋白绞链区中的一个或多个半胱氨酸残基可被一个或多个其它氨基酸残基(例如，一个或多个丝氨酸残基)所取代。或者或此外，改变的免疫球蛋白绞链区可使野生型免疫球蛋白绞链区的脯氨酸残基被另一个氨基酸残基(例如丝氨酸残基)取代。
可用作连接区的替代性铰链和接头序列可从连接IgV-样或IgC-样结构域的细胞表面受体各部分制备。当细胞表面受体含有多个串联的IgV-样结构域时在IgV-样结构域之间，以及当细胞表面受体含有多个串联的IgC-样区域时在IgC-样结构域之间的区域也可用作连接区或接头肽。在某些实施方式中，铰链和接头序列的长度是5-60个氨基酸，可能基本呈柔性，但也可能提供更刚性的特征，并可主要含有α-螺旋结构而β-片层结构很少。优选地，序列是在血浆和血清中具有稳定性，并能耐受蛋白水解切割。在一些实施方式中，序列可含有能形成一个或多个二硫键以稳定该分子C末端的天然产生的或添加的基序，如CPPC(SEQ ID NO:422)。在其它实施方式中，序列可含有一个或多个糖基化位点。铰链和接头序列的例子包括CD2、CD4、CD22、CD33、CD48、CD58、CD66、CD80、CD86、CD96、CD150、CD166和CD244的IgV样和IgC样结构域之间或IgC样或IgV样结构域之间的结构域间区域。替代性铰链也可从非免疫球蛋白超家族成员的II型受体如CD69、CD72和CD161含二硫键的区域制备。
在某些实施方式中，本发明的接头包含蝎接头。蝎接头包括衍生自免疫球蛋白超家族成员结构域间区域的肽，例如，衍生自免疫球蛋白铰链区如IgG1、IgG2、IgG3、IgG4、IgA和IgE铰链区的铰链样肽。在某些实施方式中，铰链样蝎接头保留至少一个能够在生理条件下形成链间二硫键的半胱氨酸。衍生自IgG1的蝎接头可具有1个半胱氨酸或2个半胱氨酸，并可保留对应于野生型IgG1的N末端铰链半胱氨酸的半胱氨酸。也考虑将非铰链样肽作为蝎接头，条件是这样的肽提供足够的间隔和柔性以提供能够形成两个结合域的单链蛋白质，所述各结合域相对于位置更为接近中心的恒定亚区结构域位置朝向各蛋白质末端(N和C)。示例性非铰链样蝎接头包括来自II型膜蛋白的C型凝集素柄区域的柄区域，如CD69、CD72、CD94、NKG2A和NKG2D的柄区域。在一些实施方式中，蝎接头包含选自SEQ ID NO:355-359和365的序列。
在一些实施方式中，接头具有一个用于形成链间二硫键的半胱氨酸残基。在其它实施方式中，接头具有两个用于形成链间二硫键的半胱氨酸残基。在其它实施方式中，接头衍生自免疫球蛋白结构域间区域(例如抗体绞链区)或II型C型凝集素柄区域(衍生自II型膜蛋白；参见例如，PCT申请公开号WO2007/146968所示的示例性凝集素柄区序列，如该公开文件的SEQ ID NO:111、113、115、117、119、121、123、125、127、129、131、133、135、149、151、153、155、157、159、161、163、165、167、169、231、233、235、237、239、241、243、245、247、249、251、253、255、257、259、261、263、265、267、269、271、273、275、277、279、281、287、289、297、305、307、309-311、313-331、346、373-377、380或381，这些序列通过引用纳入本文)。
在一个方面，包含本文所述CD86结合域的示例性多特异性融合蛋白还包含至少一个具有非CD86靶点特异性的额外结合区或结合域(“异源结合域”)。例如，本发明的多特异性融合蛋白具有通过间插结构域与作为IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物的结合域连接的CD86结合域。在某些实施方式中，多特异性融合蛋白包含第一和第二结合域、第一和第二接头以及间插结构域，其中间插结构域的一端通过第一接头与作为CD86结合域(例如，CTLA4胞外域、CD28胞外域、抗CD86)的第一结合域融合，另一端通过第二接头与作为IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物的不同结合域融合。
在某些实施方式中，本发明多特异性融合蛋白的第一接头和第二接头各自独立地选自例如，如SEQ ID NO:43-166所提供的接头1-133或SEQ ID NO:244、307、320、355-379和383-398所提供的接头。例如，所述第一或第二接头可以是接头47、58、126-131(分别为SEQID NO:89、100和159-164)，或SEQ ID NO:244或355-379所示接头，或其任何组合。在其它实施例中，一个接头是接头47(SEQ ID NO:89)或接头132(SEQ ID NO:165)，另一个接头是SEQID NO:355所示接头或接头127(SEQ ID NO:160)；或一个接头是接头58(SEQ ID NO:100)或接头133(SEQ ID NO:166)，另一个接头是接头126(SEQ ID NO:159)；或一个接头是接头58(SEQ ID NO:100)或接头133(SEQ ID NO:166)，另一个接头是接头127(SEQ ID NO:160)；或一个接头是接头58(SEQ ID NO:100)或接头133(SEQ ID NO:166)，另一个接头是接头128(SEQ ID NO:161)；或一个接头是接头58(SEQ ID NO:100)或接头133(SEQ ID NO:166)，另一个接头是接头129(SEQ ID NO:162)。在其它实施例中，包含如CD86特异性的VH和VL结构域的本发明结合域在VH和VL结构域之间可具有额外(第三)接头，如SEQ ID NO:244、SEQ IDNO:89所示接头，接头46(SEQ ID NO:88)、接头130(SEQ ID NO:163)或接头131(SEQ ID NO:164)。在这些实施方式的任何一个中，所述接头可在任一端(例如，接头130在(G4S)核心序列的氨基端上具有丝氨酸)或两端(例如，接头120在(G4S)核心序列的一端具有2个氨基酸(天冬酰胺-酪氨酸)，在另一端具有3个氨基酸(甘氨酸-天冬酰胺-丝氨酸))内部侧接1-5个额外氨基酸(例如，接头131具有(G4S)核心序列内部的丙氨酸)，这可能完全是形成这样的重组分子的结果(例如，采用特定限制性酶位点以连接核酸分子可导致插入一个到若干个氨基酸)，就本发明的目的而言，其可视作任何特定接头核心序列的一部分。
在其它实施方式中，本发明多特异性融合蛋白的间插结构域由免疫球蛋白恒定区或亚区组成，其中所述间插结构域位于CD86结合域和作为IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物的结合域之间。在某些实施方式中，本发明多特异性融合蛋白的间插结构域具有位于氨基末端的CD86结合域和位于羧基末端的作为IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物的结合域。在其它实施方式中，本发明多特异性融合蛋白的间插结构域具有位于氨基末端的作为IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物的结合域和位于羧基末端的CD86结合域。
在其它实施方式中，免疫球蛋白恒定区或亚区包含免疫球蛋白G1(IgG1)的CH2和CH3结构域。在相关实施方式中，所述IgG1CH2和CH3结构域具有一个或多个以下突变氨基酸(即在该位置具有不同的氨基酸)：第234位的亮氨酸(L234)、第235位的亮氨酸(L235)、第237位的甘氨酸(G237)、第318位的谷氨酸(E318)、第320位的赖氨酸(K230)、第322位的赖氨酸(K322)或其任何组合(根据Kabat编号)。例如，可将这些氨基酸中的任何一个改变为丙氨酸。在其它实施方式中，根据Kabat编号，CH2结构域中的L234、L235和G237各突变为丙氨酸(即分别为L234A、L235A和G237A)，IgG1CH3结构域中的E318、K320和K322各突变为丙氨酸(即分别为E318A、K320A和K322A)。
在某些实施方式中，本发明的多特异性融合蛋白可包含“小模块式免疫药物”(SMIPTM)。在该方面，术语SMIPTM指相对单克隆和重组抗体具有增强药物性质的高模块化化合物类别。SMIP包含具有靶点特异性结合域的单个多肽链，其基于例如抗体可变结构域，与允许特异性募集所需类别效应物细胞(如巨噬细胞和天然杀伤(NK)细胞)和/或募集补体介导的杀伤作用的可变FC区结合。根据选择的靶点和铰链区，SMIP可通过细胞表面受体进行信号传导或阻断信号传导。本文所用的称为“小模块式免疫药物”或“SMIPTM产物”的工程改造融合蛋白见美国专利公开号2003/133939、2003/0118592和2005/0136049，以及国际专利公开WO02/056910、WO2005/037989和WO2005/017148所述。
在一些实施方式中，多特异性融合蛋白可包含PIMS分子，如美国专利公开号2009/0148447和国际专利公开WO2009/023386所述分子。
在某些实施方式中，可将本发明的多特异性融合蛋白工程改造为具有不同的前端和后端亲和力以靶向特定细胞类型。例如，可采用对CD86的亲和力高于工程改造IL10激动剂(例如，具有I87A或I87S突变，或为单IL10结构)对huIL10R1亲和力的抗CD86结合域(例如，3D1、FUN1或其人源化变体)，并在本发明的交叉受体分子中组合这样的分子，以有利于靶向感兴趣的特定细胞类型，如抗原呈递细胞(APC)。在该方面，根据靶向的所需细胞类型，可制备对CD86具有较高或较低亲和力，或对本文所述任何异源靶点蛋白质具有较高或较低亲和力的融合蛋白。在优选实施方式中，CD86拮抗物结合域通过大于异源结合域对其结合伙伴亲和力的CD86亲和力优先将多靶点特异性交叉受体分子靶向APC。
在一些实施方式中，本发明的多特异性融合蛋白具有包含CTLA4胞外结构域或亚结构域、CD28胞外结构域或亚结构域、或CD86特异性抗体衍生结合域的CD86结合域。在某些实施方式中，CD86特异性抗体衍生结合域衍生自FUN1单克隆抗体(参见例如，JPathol.1993Mar；169(3):309-15)，或衍生自3D1抗CD86单克隆抗体。在某些实施方式中，CD86结合域是sCTLA4，如SEQ ID NO:1的成熟多肽序列。在某些实施方式中，CD86结合域是sCTLA4，如SEQ ID NO:1的序列，或CTLA4的可变样结构域，如SEQ ID NO:3，或其亚结构域。在某些实施方式中，CD86结合域是sCD28，如SEQ ID NO:2的成熟多肽序列(信号肽：氨基酸1-18)。在其它实施方式中，CD86结合域包含来自FUN1(例如，SEQ ID NO:305和306)或3D1(例如，SEQ ID NO:318和319)的轻链和重链可变结构域，优选scFv形式。
在其它实施方式中，本发明的多特异性融合蛋白具有CD86结合域和作为IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物的异源结合域(参见例如，SEQ ID NO：7、14、15、18-22、25、26、29、32、33、36、39和40提供的异源结合域氨基酸序列)。
这样的多特异性融合蛋白在本文称为交叉受体分子，其示例性结构包括N-BD1-ID-BD2-C和N-BD2-ID-BD1-C，其中N和C分别表示氨基末端和羧基末端；BD1是CD86结合域，如免疫球蛋白样或免疫球蛋白可变区结合域或胞外域；X是间插结构域；BD2是作为IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物的结合域。在一些构建体中，X可以包含置于第一和第二结合域之间的免疫球蛋白恒定区或亚区。在一些实施方式中，本发明的多特异性融合蛋白具有的间插结构域(X)从氨基末端到羧基末端包含以下结构：-L1-X-L2-，其中L1和L2各自独立地是含有2到约150个氨基酸的接头；X是免疫球蛋白恒定区或亚区。在其它实施方式中，所述多特异性融合蛋白具有的间插结构域是清蛋白、转铁蛋白或其它血清蛋白结合蛋白，其中所述融合蛋白在组合物中主要或充分保持单链多肽的形式。
示例性交叉受体融合蛋白的氨基酸序列见SEQ ID NO:9、13、17、24、28、31、35、38、42、171、173、175、177、179、181、183、185、187、189、191、193、195、197、199、201、203、205、207、209、211、213、215、217、219、221、223、237、239、252、254、256、258、260、262、266、276、302、330、334、336、338、340、350、352和354所示；其分别由SEQ ID NO:8、12、16、23、27、30、34、37、41、170、172、174、176、178、180、182、184、186、188、190、192、194、196、198、200、202、204、206、208、210、212、214、216、218、220、222、236、238、251、253、255、257、259、261、265、275、301、329、333、335、337、339、349、351和353所示多核苷酸序列编码。
在其它实施方式中，本发明的多特异性融合蛋白具有以下结构：N-BD1-X-L2-BD2-C，其中BD1是CD86结合域，如与CTLA4胞外域至少约90％相同的结合域；-X-是-L1-CH2CH3-，其中L1是第一IgG1铰链，任选通过取代第一或第二半胱氨酸突变，且CH2CH3-是IgG1Fc结构域的CH2CH3区；L2是选自SEQ ID NO:43-166、244、307、320、355-379或383-398的接头；BD2是如本文所述作为IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物的结合域。
在具体实施方式中，多特异性交叉受体融合蛋白具有(a)包含与SEQ ID NO:1或SEQ ID NO:2成熟多肽序列至少80％、90％、95％、96％、97％、98％、99％或至少100％相同的氨基酸序列的CD86结合域，和(b)作为IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物的结合域，其包含与SEQ ID NO:7、14、15、18-22、25、26、29、32、33、36、39和40提供的上述异源结合蛋白的相应成熟多肽序列至少80％、90％、95％、96％、97％、98％、99％或至少100％相同的氨基酸序列，其中从氨基末端到羧基末端，或从羧基末端到氨基末端，(i)(a)的CD86结合域或(b)的结合域与第一接头融合，(ii)第一接头与如SEQ ID NO:409和415-417中任何一个序列所示的免疫球蛋白重链恒定区CH2和CH3融合，(iii)CH2CH3恒定区多肽与第二接头融合，和(iv)第二接头与(a)的CD86结合域或(b)的结合域融合。在某些实施方式中，所述第一接头是接头47(SEQ ID NO:89)、接头132(SEQ ID NO:165)或接头133(SEQ ID NO:166)，所述第二接头是接头126-129(SEQ ID NO:159-162)中任何一个，CD86结合域VH和VL结构域之间的额外(第三)接头是接头130(SEQ ID NO:163)或接头131(SEQ ID NO:164)。
示例性交叉受体融合蛋白的氨基酸序列见SEQ ID NO:9、13、17、24、28、31、35、38、42、171、173、175、177、179、181、183、185、187、189、191、193、195、197、199、201、203、205、207、209、211、213、215、217、219、221、223、237、239、252、254、256、258、260、262、266、276、302、330、334、336、338、340、350、352和354所示；其分别由SEQ ID NO:8、12、16、23、27、30、34、37、41、170、172、174、176、178、180、182、184、186、188、190、192、194、196、198、200、202、204、206、208、210、212、214、216、218、220、222、236、238、251、253、255、257、259、261、265、275、301、329、333、335、337、339、349、351和353所示多核苷酸序列编码。
制备多特异性融合蛋白
为有效产生本文所述的任何结合域多肽或融合蛋白，采用前导肽促进所表达的多肽和融合蛋白的分泌。预期使用任何常规的前导肽(信号序列)以指导初始表达的多肽或融合蛋白进入分泌通路并引起从成熟多肽或融合蛋白上前导肽和所述多肽或融合蛋白之间的连接点处或其附近切割前导肽。根据本领域已知因素选择特定的前导肽，例如使用易于在前导肽的编码序列起始处或末端插入限制性内切核酸酶切割位点以便于分子工程操作的多核苷酸编码的序列，条件是这类引入序列确定的氨基酸不会不可接受地干扰由初始表达的蛋白质进行前导肽的所需处理；或如果所述前导肽在该多肽或融合蛋白成熟过程中未被切割，则其不会不可接受地干扰多肽或融合蛋白分子的任何所需功能。本发明的示例性前导肽包括天然前导序列(即随天然蛋白质一起表达的序列)或使用异源前导序列，如H3N-MDFQVQIFSFLLISASVIMSRG(X)n-CO2H，其中X是任何氨基酸，n是0-3(SEQ ID NO:167、419、420和421)，或H3N-MEAPAQLLFLLLLWLPDTTG-CO2H(SEQ ID NO:168)。
如本文所述，考虑到本文所述的结合域，如胞外域、轻链和重链可变区以及CDR的变体和衍生物。在一个实施例中，提供用一个或多个氨基酸残基补充特定结合剂氨基酸序列的插入变体。插入可位于蛋白质的任一端或两端，或可位于特定结合剂氨基酸序列的内部区域内。本发明的变体产物也包含成熟的特定结合剂产物，即去除前导或信号序列且所得蛋白质具有额外氨基末端残基的特定结合剂产物。所述额外的氨基末端残基可来自另一种蛋白质，或可包含无法鉴定为来自某具体蛋白质的一个或多个残基。考虑到在-1位上具有额外甲硫氨酸残基的多肽，以及在-2和-1位上具有额外甲硫氨酸和赖氨酸残基的本发明多肽。具有额外的Met、Met-Lys或Lys残基(或总体具有一个或多个碱性残基)的变体可特别用于提高细菌宿主细胞中的重组蛋白产量。
本文所用的“氨基酸”指天然(天然产生)的氨基酸、取代的天然氨基酸、非天然氨基酸、取代的非天然氨基酸或其任何组合。天然氨基酸的名称在本文中表示为标准的单字母或三字母代码。天然极性氨基酸包括天冬酰胺(Asp或N)和谷氨酰胺(Gln或Q)；以及碱性氨基酸如精氨酸(Arg或R)、赖氨酸(Lys或K)、组氨酸(His或H)及其衍生物；以及酸性氨基酸如天冬氨酸(Asp或D)和谷氨酸(Glu或E)及其衍生物。天然疏水性氨基酸包括色氨酸(Trp或W)、苯丙氨酸(Phe或F)、异亮氨酸(Ile或I)、亮氨酸(Leu或L)、甲硫氨酸(Met或M)、缬氨酸(Val或V)及其衍生物；以及其它非极性氨基酸如甘氨酸(GIy或G)、丙氨酸(Ala或A)、脯氨酸(Pro或P)及其衍生物。中等极性的天然氨基酸包括丝氨酸(Ser或S)、苏氨酸(Thr或T)、酪氨酸(Tyr或Y)、半胱氨酸(Cys或C)及其衍生物。除非另有说明，本文所述的任何氨基酸可以是D-或L-构型。
取代变体包括氨基酸序列中一个或多个氨基酸残基被去除或用替代性残基取代的融合蛋白。在一些实施方式中，取代具有保守性质；然而，本发明也包括非保守取代。可根据物理性质以及在蛋白质二级和三级结构中的贡献对氨基酸进行分类。本领域将保守取代认定为用具有类似性质的另一个氨基酸取代氨基酸。示例性保守取代见以下表1所示(参见WO97/09433，第10页，1997年3月13日公开)。
表1保守取代I
或者，保守氨基酸可如Lehninger(《生物化学》，第2版；纽约州沃斯出版公司(Worth Publishers，Inc.NY:NY)(1975)，第71-77页)所述分组，如以下表2所示。
表2保守取代II
变体或衍生物也可具有使用特定表达系统而产生的额外氨基酸残基。例如，使用将所需多肽表达为谷胱甘肽-S-转移酶(GST)融合产物一部分的市售载体能提供在切割所需多肽的GST组分后在-1位上具有额外甘氨酸残基的所需多肽。也考虑到在其它载体系统中表达产生的变体，包括将组氨酸标签引入氨基酸序列，通常位于序列的羧基和/或氨基末端的变体。
也考虑到去除本发明结合域中一个或多个氨基酸残基的缺失变体。缺失可以在融合蛋白的一端或两端实现，或通过去除氨基酸序列内的一个或多个残基进行。
在某些示例性实施方式中，本发明的融合蛋白为糖基化形式，糖基化模式取决于各种因素，包括表达蛋白质的宿主细胞(如果在重组宿主细胞中制备)和培养条件。
本发明也提供融合蛋白的衍生物。衍生物包括携带氨基酸残基插入、缺失或取代以外的修饰的特异性结合域多肽。在某些实施方式中，修饰是共价修饰，包括例如，与聚合物、脂质、其它有机和无机部分发生化学键合。可制备本发明的衍生物以增加特异性结合域多肽的循环半衰期，或可设计所述衍生物以提高所述多肽对所需细胞、组织或器官的靶向容量。
本发明还包括经共价修饰或衍生化而包含一个或多个水溶性聚合物连接体如聚乙二醇、聚氧乙烯二醇或聚丙二醇的融合蛋白，如美国专利号4,640,835、4,496,689、4,301,144、4,670,417、4,791,192和4,179,337所述。本领域已知的其它有用的聚合物包括单甲氧基-聚乙二醇、葡聚糖、纤维素和其它基于碳水化合物的聚合物、聚(N-乙烯基吡咯烷酮)-聚乙二醇、丙二醇均聚物、聚环氧丙烷/环氧乙烷共聚物、聚氧乙烯化多元醇(例如甘油)和聚乙烯醇，以及这些聚合物的混合物。特别优选聚乙二醇(PEG)衍生的蛋白质。水溶性聚合物可在特定位置键合，例如在本发明蛋白质或多肽的氨基末端，或与所述多肽的一个或多个侧链随机连接。使用PEG提高治疗能力见美国专利号6,133,426所述。
本发明的具体实施方式是免疫球蛋白或Fc融合蛋白。这类融合蛋白可具有较长的半衰期，例如，数小时、一天或更长时间，或一周或更长时间，特别是在Fc结构域能够与FcRn，即新生Fc受体相互作用时。Fc结构域中与FcRn的结合位点也是细菌蛋白A和G结合的位点。这些蛋白质之间的紧密结合可用作纯化本发明抗体或融合蛋白的手段，例如，在蛋白质纯化过程中使用蛋白A或蛋白G亲和色谱。
蛋白质纯化技术是本领域技术人员众所周知的。这些技术包括在某一水平上对多肽和非多肽组分进行粗分级。常常需要使用色谱和电泳技术进行进一步纯化，以实现部分或完全纯化(或纯化至均一)。特别适合制备纯融合蛋白的分析方法是离子交换色谱、排阻色谱、聚丙烯酰胺凝胶电泳和等电聚焦。特别有效的肽纯化方法是快速蛋白质液相色谱和HPLC。
本发明的某些方面涉及融合蛋白的纯化，在具体实施方式中，涉及其充分纯化。本文所用术语“纯化的融合蛋白”指可与其它组分分离的组合物，其中融合蛋白相对于其天然可获得状态纯化至任何程度。因此，纯化的融合蛋白也指与其天然产生环境相分离的融合蛋白。
通常，“纯化”指经过分级去除各种其它组分的融合蛋白组合物，所述组合物基本保持其表达的生物学活性。在使用术语“基本纯化”时，该说明指融合结合蛋白组合物，其中融合蛋白构成该组合物的主要组分，例如占组合物中蛋白质重量的约50％、约60％、约70％、约80％、约90％、约95％、约99％或更多。
本领域技术人员能够从本发明中获知定量分析纯化程度的各种方法。这些方法包括例如，测定活性组分的特异性结合活性，或通过SDS/PAGE分析评价组分中融合蛋白的含量。评价蛋白质组分纯度的优选方法是计算该组分的结合活性，将其与初始提取物的结合活性作比较，从而计算纯化程度，在本文中通过“纯化倍数”评价。当然，用于表示结合活性水平的实际单位取决于选择用于跟踪纯化的具体鉴定技术和表达的融合蛋白是否显示可检测的结合活性。
适用于蛋白质纯化的各种技术是本领域技术人员众所周知的。这些技术包括例如，用硫酸铵、PEG、抗体等沉淀，或通过先热变性再离心进行沉淀；色谱步骤如离子交换、凝胶过滤、反相、羟基磷灰石和亲和色谱；等电聚焦；凝胶电泳；以及这些技术和其它技术的组合。正如本领域众所周知的那样，认为可改变进行各种纯化步骤的顺序，或可省略某些步骤，并仍产生适合制备基本纯化的蛋白质的方法。
通常并不要求总是以最纯化状态提供融合蛋白。实际上，考虑到在某些实施方式中可使用纯化水平较低的蛋白质。可结合使用较少的纯化步骤，或利用同一总纯化方案的不同形式达到部分纯化。例如，应理解利用HPLC设备进行的阳离子交换柱色谱的纯化水平通常高于利用低压色谱系统的相同技术的纯化水平。显示较低相对纯化程度的方法可能在蛋白质产物总回收率方面，或在维持所表达蛋白质的结合活性方面具有优点。
已知多肽迁移可随不同的SDS/PAGE条件发生改变，有时是显著改变(Capaldi等(1977)Biochem.Biophys.Res.Comm.76:425)。因此，应理解在不同的电泳条件下，纯化或部分纯化的融合蛋白表达产物的表观分子量可能不同。
多核苷酸、表达载体和宿主细胞
本发明提供编码本发明多特异性融合蛋白的多核苷酸(分离或纯化或纯的多核苷酸)、包含这类多核苷酸的载体(包括克隆载体和表达载体)以及用本发明多核苷酸或载体转化或转染的细胞(例如宿主细胞)。
在某些实施方式中，考虑到编码本发明结合域，或含有一个或多个这类结合域的多特异性融合蛋白的多核苷酸(DNA或RNA)。所附实施例中提供了编码多特异性融合蛋白构建体的表达盒。
本发明还涉及包含本发明多核苷酸的载体，具体为重组表达构建体。在一个实施方式中，本发明考虑到包含编码一种多特异性融合蛋白的多核苷酸的载体，所述多特异性融合蛋白包含本发明的CD86结合域和IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物结构域，以及引起或促进这样的多特异性融合蛋白编码序列的转录、翻译和加工的其它多核苷酸序列。
用于原核和真核宿主的合适的克隆和表达载体见例如，Sambrook等，《分子克隆：实验室手册(Molecular Cloning:A Laboratory Manual)》，第2版，冷泉港(Cold SpringHarbor)，纽约州，(1989)所述。示例性的克隆/表达载体包括可基于质粒、噬菌粒、噬粒、粘粒、病毒、人工染色体或本领域已知适用于扩增、转移和/或表达所含多核苷酸的任何核酸载体的克隆载体、穿梭载体和表达构建体。
本文所用的“载体”表示能够运输所连接的另一核酸的核酸分子。示例性载体包括质粒、酵母人工染色体和病毒基因组。某些载体可在宿主细胞中自主复制，而另一些载体可整合到宿主细胞基因组中从而随宿主基因组一起复制。此外，某些载体在本文中称为“重组表达载体”(或简称为“表达载体”)，其含有操作性连接于表达控制序列并因此能够指导这些序列表达的核酸序列。
在某些实施方式中，表达构建体衍生自质粒载体。说明性构建体包括修饰的pNASS载体(加利福尼亚州帕洛阿尔托的克隆泰克公司(Clontech,Palo Alto,CA))，其具有编码氨苄青霉素抗性基因、多腺苷酸化信号和T7启动子位点的核酸序列；pDEF38和pNEF38(CMCICOS生物技术公司(CMC ICOS Biologics，Inc.))，其具有CHEF1启动子；和pD18(隆萨公司(Lonza))，其具有CMV启动子。其它合适的哺乳动物表达载体是众所周知的(参见例如，Ausubel等，1995；Sambrook等，同上；也参见例如，加利福尼亚州圣迭戈的英杰公司(Invitrogen,San Diego,CA)、威斯康星州麦迪逊的诺华基公司(Novagen,Madison,WI)、新泽西州皮斯卡塔维的法玛西亚公司(Pharmacia,Piscataway,NJ)的产品目录)。可制备的有用构建体包含位于合适调控下的二氢叶酸还原酶(DHFR)编码序列，用来促进融合蛋白产量水平提高，所述产量水平取决于施加合适选择剂(如氨甲蝶呤)后的基因扩增。
通常，重组表达载体包含允许转化宿主细胞的复制起点和选择性标记，以及衍生自高表达基因的指导下游结构序列转录的启动子，如上所述。与本发明多核苷酸操作性连接的载体产生克隆或表达构建体。示例性的克隆/表达构建体含有至少一个与本发明多核苷酸操作性连接的表达控制元件，例如启动子。也考虑在载体和本发明克隆/表达构建体中采用其它表达控制元件，如增强子、因子特异性结合位点、终止子和核糖体结合位点。本发明多核苷酸的异源结构序列以合适状态与翻译起始和终止序列装配在一起。因此，例如，本文提供的融合蛋白编码核酸可包含在任何表达载体构建体中，形成用于在宿主细胞中表达这类蛋白质的重组表达构建物。
可例如通过各种方法将合适的DNA序列插入载体中。通常，通过本领域已知方法将DNA序列插入合适的限制性核酸内切酶切割位点。考虑用于克隆、DNA分离、扩增和纯化的标准技术，用于涉及DNA连接酶、DNA聚合酶、限制性内切核酸酶等的酶促反应的标准技术以及各种分离技术。多种标准技术见例如，Ausubel等(《新编分子生物学实验指南(CurrentProtocols in Molecular Biology)》，马萨诸塞州波士顿的格林出版联合公司和约翰韦利森公司(Greene Publ.Assoc.Inc.&John Wiley&Sons，Inc.，Boston，MA)，1993)；Sambrook等(《分子克隆(Molecular Cloning)》，第2版，纽约州普莱恩维尤的冷泉港实验室出版社(Cold Spring Harbor Laboratory，Plainview，NY)，1989)；Maniatis等(《分子克隆》，纽约州普莱恩维尤的冷泉港实验室出版社，1982)；Glover(编)(《DNA克隆(DNA Cloning)》第I卷和第II卷，英国牛津的IRL出版社(IRL Press，Oxford，UK)，1985)；Hames和Higgins(编)(《核酸杂交(Nucleic Acid Hybridization)》，英国牛津的IRL出版社，1985)；等等。
表达载体中的DNA序列与至少一个合适的表达控制序列(例如组成性启动子或调节性启动子)操作性连接，以指导mRNA合成。这类表达控制序列的代表性例子包括真核细胞或其病毒的启动子，如上所述。可利用CAT(氯霉素转移酶)载体或具有选择性标记的其它载体从任何所需基因中选择启动子区域。真核启动子包括CMV立即早期、HSV胸苷激酶、早期和晚期SV40、逆转录病毒的LTR和小鼠金属硫蛋白-I。本领域普通技术人员熟知如何选择合适的载体和启动子，本文描述了某些特别优选的包含与编码本发明蛋白质或多肽的核酸操作性连接的至少一个启动子或调节性启动子的重组表达构建体的制备。
也考虑了本发明多核苷酸的变体。变体多核苷酸与本文所述确定序列的多核苷酸之一至少90％相同，优选95％、99％或99.9％相同，或能够在严格杂交条件下与确定序列的多核苷酸之一杂交，所述严格杂交条件为0.015M氯化钠、0.0015M柠檬酸钠，约65-68℃或0.015M氯化钠、0.0015M柠檬酸钠和50％甲酰胺，约42℃。多核苷酸变体保持编码具有本文所述功能的结合域或其融合蛋白的能力。
术语“严格”用于指本领域通常理解的严格条件。杂交严格性通常取决于温度、离子强度和变性剂如甲酰胺的浓度。用于杂交和洗涤的严格条件的例子是0.015M氯化钠、0.0015M柠檬酸钠，约65-68℃；或者0.015M氯化钠、0.0015M柠檬酸钠和50％甲酰胺，约42℃(参见Sambrook等，《分子克隆：实验室手册(Molecular Cloning:A Laboratory Manual)》，第2版，纽约州冷泉港的冷泉港实验室出版社(Cold Spring Harbor Laboratory，ColdSpring Harbor，N.Y.)，1989)。
也可采用更严格的条件(如更高的温度、更低的离子强度、更多的甲酰胺或其它变性剂)；然而，杂交速率会受影响。在涉及脱氧寡核苷酸杂交的情况中，其它示例性严格杂交条件包括在37℃(用于14个碱基的寡核苷酸)、48℃(用于17个碱基的寡核苷酸)、55℃(用于20个碱基的寡核苷酸)和60℃(用于23个碱基的寡核苷酸)下，用6x SSC、0.05％焦磷酸钠洗涤。
本发明的另一方面提供用本发明的任何多核苷酸或载体/表达构建体转化或转染的或含有本发明的任何多核苷酸或载体/表达构建物的宿主细胞。利用本领域已知的任何方法，包括转化、转染和转导，将本发明的多核苷酸或克隆/表达构建体引入合适的细胞。宿主细胞包括经历离体细胞治疗，包括例如，离体基因治疗的对象的细胞。携带本发明多核苷酸、载体或蛋白质时，作为本发明某一方面考虑的真核宿主细胞除对象自身细胞(例如人患者的自身细胞)外还包括VERO细胞、HeLa细胞、中华仓鼠卵巢(CHO)细胞系(包括能够修饰所表达多价结合分子的糖基化模式的修饰的CHO细胞，参见美国专利申请公开号2003/0115614)、COS细胞(如COS-7)、W138、BHK、HepG2、3T3、RIN、MDCK、A549、PC12、K562、HEK293细胞、HepG2细胞、N细胞、3T3细胞、草地贪夜蛾(Spodoptera frugiperda)细胞(例如Sf9细胞)、酿酒酵母(Saccharomyces cerevisiae)细胞以及本领域已知可用于表达和任选地分离本发明蛋白质或肽的任何其它真核细胞。还考虑了原核细胞，包括大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、鼠伤寒沙门氏菌(Salmonellatyphimurium)、链霉菌、或者本领域已知适用于表达和任选地分离本发明蛋白质或肽的任何原核细胞。具体而言，在从原核细胞分离蛋白质或肽时，考虑可采用本领域已知的用于从内含体提取蛋白质的技术。本领域技术人员根据本文教导能够了解如何选择合适的宿主。考虑到糖基化本发明融合蛋白的宿主细胞。
术语“重组宿主细胞”(或简称为“宿主细胞”)指含有重组表达载体的细胞。应理解，这类术语不仅指特定对象细胞，还指该细胞的后代。由于在连续传代过程中可能因突变或环境影响而发生某些改变，因此这类后代实际上可能不与亲代细胞完全相同，但仍落入本文所用术语“宿主细胞”的范围内。
可在经修饰适合激活启动子、选择转化体或扩增特定基因的常规营养培养基中培养重组宿主细胞。本领域普通技术人员不难了解选择用于表达的特定宿主细胞的培养条件，如温度、pH等。也可利用各种哺乳动物细胞培养系统来表达重组蛋白。哺乳动物表达系统的例子包括如Gluzman(1981)Cell 23:175所述的猴肾成纤维细胞的COS-7细胞系，以及能够表达相容性载体的其它细胞系，例如C127、3T3、CHO、HeLa和BHK细胞系。哺乳动物表达载体包含复制起点、合适的启动子和任选的增强子，还包含任何必需的核糖体结合位点、多腺苷酸化位点、剪接供体和受体位点、转录终止序列和5′-侧接的非转录序列，例如，本文中关于多价结合蛋白表达构建体的制备中所述。衍生自SV40剪接的DNA序列和多腺苷酸化位点可用于提供所需的非转录遗传元件。可通过本领域技术人员熟悉的各种方法包括磷酸钙转染、DEAE-葡聚糖介导的转染或电穿孔将所述构建体引入宿主细胞(Davis等(1986)《分子生物学基本方法(Basic Methods in Molecular Biology)》)。
在一个实施方式中，通过指导本发明蛋白质或多肽的重组病毒构建体转导宿主细胞。转导的宿主细胞能产生含有所表达的蛋白质或多肽的病毒颗粒，所表达的蛋白质或多肽来自病毒出芽期间引入病毒颗粒的宿主细胞膜的部分。
组合物及其使用方法
为治疗患有与CD86、IL10、HLA-G、IL-35、PD-1、BTLA、LIGHT、GITRL或CD40调节异常有关的疾病状况的人或非人哺乳动物，按照一次或多次给药的方案将本发明多特异性融合蛋白以有效改善该疾病状况的症状的量给予对象。作为多肽，本发明多特异性融合蛋白可悬浮于或溶解于药学上可接受的稀释剂，其任选包含其它药学上可接受的赋形剂的稳定剂，可用于静脉内注射或输注给药，如下文所详述。
药学有效剂量是预防、抑制疾病状况的发生或治疗疾病状态(在某种程度上缓解其症状，优选所有症状)的剂量。药学有效剂量取决于疾病类型、所用组合物、给药途径、所治疗对象的类型、考虑治疗的特定对象的身体特征、平行用药和医学领域技术人员应认识到的其它因素。例如，可根据本发明的结合域多肽或多特异性蛋白融合物的效力，给予0.1mg/kg至100mg/kg体重的活性成分(可作为单次剂量给予，或作为多次剂量每小时、每天、每周、每月给予一次，或以合适间隔组合给予)。
在某些方面，本发明提供融合蛋白的组合物。本发明的药物组合物通常包含一种或多种类型的结合域或融合蛋白，结合药学上可接受的载体、赋形剂或稀释剂。这类载体在所用剂量和浓度下应对接受者无毒。用于治疗应用的药学上可接受的载体是药学领域众所周知的，见例如《雷明顿药物科学(Remington’s Pharmaceutical Sciences)》(麦克出版公司(Mack Publishing Co.),A.R.Gennaro编，1985)。例如，可采用生理pH的无菌盐水和磷酸缓冲盐水。药物组合物中可提供防腐剂、稳定剂、染料等。例如，可添加苯甲酸钠、山梨酸或对羟基苯甲酸酯作为防腐剂，同上，1449。此外，也可采用抗氧化剂和悬浮剂，同上。本发明的化合物可以游离碱或盐形式使用，认为这两种形式均落入本发明的范围内。
药物组合物还可含有稀释剂如缓冲液，抗氧化剂如抗坏血酸，低分子量(小于约10个残基)多肽，蛋白质，氨基酸，碳水化合物(例如葡萄糖、蔗糖或糊精)，螯合剂(例如EDTA)，谷胱甘肽，或其它稳定剂或赋形剂。中性缓冲盐水或混有非特异性血清白蛋白的盐水是示例性的合适稀释剂。优选采用合适的赋形剂溶液作为稀释剂，将产物配制成冻干物。
本发明的组合物可用于治疗人和非人哺乳动物中由CD86、IL-10、HLA-G、IL-35、PD-1、BTLA、LIGHT、GITRL或CD40调节异常引起的或与其相关的疾病状况。如上所述，已显示通过例如给予CTLA4Ig阻断CD86与CD28结合在治疗自身免疫疾病如类风湿性关节炎方面具有有效性。已知IL10具有免疫抑制性质(Commins等(2008)J.Allergy Clin.Immunol.121:1108-11；Ming等(2008)Immunity 28:468-476)，向牛皮癣(Asadullah等(1999)Arch.Dermatol.135:187-92)和炎性肠病(Schreiber等(2000)Gastroenterology 119:1461-72)患者给予IL10后观察到有益反应。如上所述，已表明HLA-G可用于降低CNS中与多发性硬化相关的炎症应答(Wiendl等(2005)Blood,128:2689-2704)，并作为促进移植中移植物耐受性的治疗剂(Carosella等(2008)Blood 111:4862-4870)。已显示HGF可在关节炎小鼠模型和哮喘小鼠模型中有效减轻疾病。已显示IL35可在关节炎小鼠模型中有效减轻疾病并抑制T细胞增殖。如上所述，已显示LIGHT拮抗物可有效减轻移植物抗宿主疾病并抑制T细胞增殖。此外，认为LIGHT在炎性肠病和克罗恩病(Crohn’s disease)中发挥作用。已显示PD1-L1或PD1-L2与PD-1的结合可有效减少T细胞激活和细胞因子产生。已显示BTLA可有效减少T细胞激活和细胞因子产生。已显示GITRL与GITR的结合可增大哮喘和关节炎动物模型中的疾病严重程度，并已知其增加T细胞炎症和免疫应答。如上所述，CD40信号传导参与疾病如自身免疫疾病、癌症、器官和组织移植排斥。
因此，本发明的多特异性融合蛋白可用于治疗各种自身免疫疾病和/或炎性疾病，如类风湿性关节炎、青少年类风湿性关节炎、哮喘、系统性红斑狼疮(SLE)、炎性肠病(包括克罗恩病和溃疡性结肠炎)、移植物抗宿主疾病、牛皮癣、多发性硬化、皮肌炎、多肌炎、恶性贫血、原发性胆汁性肝硬变、急性播散性脑脊髓炎(ADEM)、阿狄森病(Addison’s disease)、强直性脊柱炎、抗磷脂抗体综合征(APS)自身免疫肝炎、1型糖尿病、古德帕斯彻综合征(Goodpasture's syndrome)、格雷夫斯病(Grave’s disease)、格-巴综合征(Guillain-Barrésyndrome)(GBS)、桥本病(Hashimoto’s disease)、特发性血小板减少性紫癜、红斑狼疮、寻常天疱疮、斯耶格伦综合征( syndrome)、颞动脉炎(也称为“巨细胞性动脉炎”)、自身免疫性溶血性贫血、大疱性类天疱疮、脉管炎、腹部疾病、子宫内膜异位症、化脓性汗腺炎、间质性膀胱炎、局限性硬皮病、硬皮病、发作性睡病、神经肌强直、白斑病和自身免疫性内耳病。此外，本发明的多特异性融合蛋白可用于抑制器官移植(包括实体器官移植或同种异体移植)、细胞移植或类似移植中有害的免疫对抗反应。
“药学上可接受的盐”指药学上可接受且具有母体化合物的所需药理活性的本发明结合域多肽或融合蛋白的盐。这类盐包括以下：(1)与以下酸形成的酸加成盐：无机酸如盐酸、氢溴酸、硫酸、硝酸、磷酸等；或有机酸如乙酸、丙酸、己酸、环戊烷丙酸、乙醇酸、丙酮酸、乳酸、丙二酸、琥珀酸、苹果酸、马来酸、延胡索酸、酒石酸、柠檬酸、苯甲酸、3-(4-羟基苯甲酰)苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、1,2-乙二磺酸、2-羟基乙磺酸、苯磺酸、4-氯苯磺酸、2-萘磺酸、4-甲苯磺酸、樟脑磺酸、4-甲基双环[2.2.2]-辛-2-烯-1-羧酸、葡庚糖酸、3-苯基丙酸、三甲基乙酸、叔丁基乙酸、月桂基硫酸、葡糖酸、谷氨酸、羟基萘酸、水杨酸、硬脂酸、粘康酸等形成；或(2)母体化合物中存在的酸性质子被金属离子，如碱金属离子、碱土金属离子或铝离子取代时，或与有机碱如乙醇胺、二乙醇胺、三乙醇胺、N-甲基葡糖胺等形成配位化合物形成的盐。
在具体的说明性实施方式中，通过(例如)弹丸注射或输注静脉内给予本发明的多肽或融合蛋白。除静脉内给药外，给药途径还包括口服、局部、胃肠道外(例如舌下或口颊)、舌下、直肠、阴道和鼻内途径。本文所用术语“胃肠道外”包括皮下注射、静脉内、肌内、胸骨内、海绵体内、鞘内、耳道内、尿道内注射或输注技术。配制药物组合物以使该组合物给予患者后其中所含的活性成分可被生物利用。给予患者的组合物可采取一个或多个剂量单位的形式，例如，片剂可以是单一剂量单位，气溶胶形式的一种或多种本发明化合物的容器中可包含多个剂量单位。
就口服给药而言，可存在赋形剂和/或粘合剂，如蔗糖、高岭土、甘油、淀粉葡聚糖、环糊精、藻酸钠、乙基纤维素和羧甲基纤维素。可任选存在甜味剂、防腐剂、染料/着色剂、风味增强剂或其任何组合。还可任选使用包衣壳。
在用于进行注射给药的组合物中，可任选包含表面活性剂、防腐剂、润湿剂、分散剂、悬浮剂、缓冲液、稳定剂、等张剂中的一种或多种或其任何组合。
就基于核酸的制剂或包含本发明表达产物的制剂而言，通过例如皮内、皮下、肌内或静脉内途径，或本领域已知适合在给定情况下使用的任何途径给予约0.01μg/kg到约100mg/kg体重的剂量。μ例如，优选剂量为约1μg/kg到约20mg/kg，特别优选约5μg/kg到约10mg/kg。本领域技术人员可明显看出，给药的次数和频率取决于宿主的反应。
本发明的药物组合物可采用能够给予患者的任何形式，例如固体、液体或气体(气溶胶)形式。所述组合物可以是通过本文所述的任何途径给予的液体形式，如酏剂、糖浆、溶液、乳液或悬浮液。
本文所用的液体药物组合物，无论采用溶液、悬浮液或其它类似形式，均可包含一种或多种以下组分：无菌稀释剂如注射用水、盐水溶液(例如生理盐水)、林格溶液、等张氯化钠、不挥发性油如可用作溶剂或悬浮介质的合成的甘油单酯或甘油二酯、聚乙二醇、甘油、丙二醇或其它溶剂；抗菌剂如苄醇或对羟基苯甲酸甲酯；抗氧化剂如抗坏血酸或亚硫酸氢钠；缓冲剂如乙酸盐、柠檬酸盐或磷酸盐；螯合剂如乙二胺四乙酸；和调节张力的试剂如氯化钠或葡聚糖。胃肠道外制剂可封装在安瓿、一次性注射器或玻璃或塑料制成的多剂量小瓶中。生理盐水是优选的添加剂。可注射药物组合物优选无菌。
制剂中也可能需要包含其它组分，如递送载体，包括铝盐、油包水乳液、生物可降解的油载体、水包油乳液、生物可降解的微胶囊和脂质体。用于这类载体的佐剂的例子包括N-乙酰胞壁酰-L-丙氨酸-D-异谷氨酰胺(MDP)、脂多糖(LPS)、葡聚糖、IL-12、GM-CSF、γ-干扰素和IL-15。
尽管本领域普通技术人员已知的任何合适载体均可用于本发明的药物组合物，但可根据给药方式和是否需要缓释而改变载体类型。就胃肠道外给药而言，载体可包含水、盐水、醇、脂肪、蜡、缓冲剂或其任何组合。就口服给药而言，可采用任何上述载体或固体载体，如甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、滑石、纤维素、葡萄糖、蔗糖、碳酸镁或其任何组合。
也考虑将本发明的多特异性融合蛋白组合物与第二试剂联合给药。第二试剂可以是本领域接受的作为特定疾病状态，如炎症、自身免疫病和癌症的标准治疗的试剂。考虑到的示例性第二试剂包括细胞因子、生长因子、类固醇、NSAID、DMARD、化疗疗法、放疗疗法或其它活性和辅助性试剂。
本发明考虑到包含本发明药物组合物的剂量单位。这样的剂量单位包括例如，单剂量或多剂量小瓶或注射器，包括两室小瓶或注射器，一室包含冻干形式的本发明药物组合物，另一室包含用于重建的稀释剂。多剂量的剂量单位也可以是例如，用于连接静脉内输注装置的袋或管。
本发明也考虑到在单位剂量或多剂量容器如小瓶中包含药物组合物，以及将所述组合物给予本文所述疾病患者的一组说明的试剂盒。
本说明书中提及的所有美国专利、美国专利申请公开、美国专利申请、外国专利、外国专利申请、非专利出版物、表格、序列、网页等均通过引用整体纳入本文。以下实施例用于说明而非限制本发明。
实施例
交叉受体序列
具有CTLA4胞外域的示例性多特异性融合蛋白的核苷酸表达盒和氨基酸序列分别见SEQ ID NO:8、12、16、23、27、30、34、37和41以及SEQ ID NO:9、13、17、24、28、31、35、38和42所示。这些示例性多特异性融合蛋白的活性如以下实施例1-6所述进行测试。以下实施例中所用的缩写包括以下术语：PBS-T:PBS，pH 7.2-7.4和0.1％20；工作缓冲液:含1％BSA的PBS-T；封闭缓冲液:含3％BSA的PBS-T。
实施例1
抗CD86结合域
采用杂交瘤3D1和FUN1克隆这些单克隆抗体的抗CD86可变结合域。来自FUN1和3D1抗CD86单克隆抗体的重链、轻链、scFv接头和CDR的序列分别见SEQ NO:305-313和318-326所示。
采用以下人源化FUN1抗CD86单克隆抗体可变结合域构建SMIP蛋白和交叉受体。如下所述将FUN1CDR移植到人种系序列中：(1)FUN1-11具有用于轻链的Igkv4-1*01FR和用于重链的IgHV1-F*01FR；(2)FUN1-21具有用于轻链的Igkv4-1*01FR和用于重链的IgHV1-2*02FR；(3)FUN1-31具有用于轻链的Igkv4-1*01FR和用于重链的IgHV3-11*01FR；(4)FUN1-12具有用于轻链的Igkv1-27*01FR和用于重链的IgHV1-F*01FR；(5)FUN1-22具有用于轻链的Igkv1-27*01FR和用于重链的IgHV1-2*02FR；和(6)FUN1-32具有用于轻链的Igkv1-27*01FR和用于重链的IgHV3-11*01FR。人源化分子的种系CDR与原始FUN1分子类似。
因此，还产生如下6种形式的人源化FUN1SMIP蛋白：(1)FUN1-11(SEQ ID NO:225)；(2)FUN1-21(SEQ ID NO:227)；(3)FUN1-31(SEQ ID NO:229)；(4)FUN1-12(SEQ ID NO:231)；(5)FUN1-22(SEQ ID NO:233)；和(6)FUN1-32(SEQ ID NO:235)。这些人源化FUN1SMIP分子的结合活性见图7所示。此外，采用FUN1可变结构域(scFv)和人源化FUN1scFv制备交叉受体，如IL10::FUN1(SEQ ID NO:183)、FUN1::IL10(SEQ ID NO:187)、FUN1-21::IL10(SEQID NO:237)、IL10::FUN1-21(SEQ ID NO:254)、IL10I87A::FUN1-21(SEQ ID NO:258)，还采用短A2铰链(SEQ ID NO:276)制备单IL10::FUN1-21结合域(其中所述A2铰链氨基酸序列见SEQ ID NO:364所示)。
将这些和本文所述的所有其它构建体均克隆到合适的哺乳动物表达载体中并在各种细胞系中表达，以产生用于特定功能鉴定的蛋白质。
实施例2
通过BIAcoreTM检测交叉受体与IL10-R1结合
检测包含CTLA4胞外域和IL10结构域的交叉受体(SEQ ID NO:9)的IL10-R1结合活性，基本如下所述。
采用HBS-P+(GE医疗保健公司(GE Healthcare))作为运行缓冲液在BIAcoreTMT100SPR(厄普萨拉的法玛西亚生物技术公司(Pharmacia Biotech AB,Uppsala))上进行表面等离振子共振(SPR)测定。采用标准胺偶联化学(GE医疗保健公司的Biacore胺偶联试剂盒)将IL-10R1(25μg/mL溶于10mM醋酸钠，pH 4.0；R&D系统公司(R&D Systems))直接固定到CM5芯片上，最终固定水平为867、2687和6719Ru(共振单位)。以50μl/分钟的流速、100pM-10nM的系列浓度注射含IL-10的构建体300秒。监测解离1200秒，通过注射pH 7.58的2M氯化镁60秒，之后注射20mM EDTA(溶于HBS-P+)60秒，来再生表面。与表面的结合相互作用在至少30个再生循环中具有稳定性。采用T100的2.0版Bia评价(BiaEvaluation)软件(GE医疗保健公司)来分析数据。CTLA4/IL10交叉受体与固定化IL-10R1的结合动力学无法用1:1朗缪尔(Langmuir)结合模型拟合，但可以高精确度拟合至二价分析物结合模型。可通过使观察到的饱和反应拟合至稳态平衡模型来以高精确度计算各构建体的平衡解离常数(KD)，见以下表3所示。将CTLA4胞外域引入交叉受体融合蛋白对IL10/IL10R1相互作用无明显影响。
表3
*文献值(Yoon等(2006)J.Biol.Chem 281:35088-35096)
**从ka1和kd1计算得出
实施例3
通过ELISA检测交叉受体结合CD80以及同时结合CD80和IL10
通过ELISA检测称为CTLA4-N2的CTLA4-Ig构建体阿巴西普(SEQ ID NO:10和11)和SEQ ID NO:9的CTLA4/IL10交叉受体的CD80和IL10R的结合活性，基本如下所述。
CD80结合
用CD80-mIg(安赛尔公司(Ancell)，目录号510-020)的2μg/ml溶液涂覆96孔黑Maxisorp CD80板(纽恩克公司(Nunc)，目录号437111)各孔，并在4℃温育过夜。然后用封闭缓冲液(含3％脱脂干牛奶的PBS-T)封闭所述板。将用封闭缓冲液连续稀释的待测试蛋白质样品加入CD80-mIg涂覆板的双孔中，覆盖所述板，在室温温育约1小时。洗涤后，每孔加入100μl用封闭缓冲液1:1000稀释的辣根过氧化物酶山羊抗人IgG(γ)，覆盖所述板，在室温温育60分钟，之后用QuantaBluTM发荧光过氧化物酶底物(热科学公司(ThermoScientific)，目录号15169)在室温温育10分钟。在420nm读出各孔的吸光度。将不相关融合蛋白TRU-015用作阴性对照。
这些实验的结果见图1所示。发现CTLA4-N2以及阿巴西普以该ELISA形式结合CD80-mIg，而CTLA4/IL10交叉受体看来显示较弱的CD80结合。
交叉受体与sIL10R1和sCD80二者结合
用sIL10Ra(R&D系统公司，目录号510-020)的2μg/ml溶液涂覆96孔黑MaxisorpCD80板(纽恩克公司，目录号437111)各孔，并在4℃温育过夜。然后用封闭缓冲液(含3％脱脂干牛奶的PBS-T)封闭所述板。将用封闭缓冲液连续稀释的待测试蛋白质样品加入sIL10Ra涂覆板的双孔中，覆盖所述板，在室温温育约1小时。洗涤后，加入10ng/μl的抗CD152抗体(安赛尔公司#359-020)或5μg/ml的CD80-mIg(安赛尔公司#510-020)，之后加入用封闭缓冲液1:1000稀释的辣根过氧化物酶山羊抗小鼠IgG(Fc)(皮尔斯公司(Pierce)#31439)，覆盖所述板，在室温温育60分钟，之后用QuantaBluTM发荧光过氧化物酶底物(热科学公司，目录号15169)在室温温育10分钟。在420nm读出各孔的吸光度。将不相关融合蛋白TRU-015用作阴性对照。
图2显示获自CTLA4-N2和SEQ ID NO:9的CTLA4::IL10交叉受体的结果。这些结果显示CTLA4-Ig-IL10交叉受体的CTLA4和IL10结构域二者能够与其配体/受体同时结合。
实施例4
交叉受体诱导的STAT3磷酸化
已知IL10与IL10-R1结合可激活Jak-1和Tyk，其进而引起STAT3的激活(参见例如，Williams等(2007)J.Biol.Chem.282:6965-6975)。此外，研究表明，可采用流式细胞术研究PBMC中STAT3的磷酸化(Lafarge等(2007)BMC Mol.Biol.8:64)。基本如下所述检测包括SEQID NO:9的CTLA4/IL10交叉受体在内的各种含IL10构建体诱导人PBMC中STAT3磷酸化的能力。
从人供体分离PBMC，并在完全培养基(RPMI，10％FBS，青霉素/链霉素)中以2x106个细胞/毫升的密度培养过夜。第二天早上将PBMC洗涤一次，用预热的RPMI 1640(无补充物)重悬达到4x106个细胞/毫升的密度，在37℃温育2.5小时。以2X浓度在0.25mL RPMI1640中进行处理，与1x106个PBMC在0.25mL RPMI 1640中混合。然后将所述样品在37℃温育15分钟。完成15分钟温育之后，向各管加入冰冷的BD Cytofix缓冲液(BD生物科学公司(BDBiosciences)，目录号554655)。将细胞在冰上培育30分钟，然后用2ml DPBS+2.5％FBS(FACS缓冲液)洗涤。倾析并涡旋样品之后，向各管加入0.5mL冰冷的BD PERM缓冲液III(BD生物科学公司，目录号558050)，然后将所述样品在冰上培育30分钟。用2mL FACS缓冲液将样品洗涤3次，最后一次洗涤后将其重悬在约0.2mL FACS缓冲液中。向各样品加入20μl偶联FITC的抗人STAT3mAb(BD生物科学公司，克隆PY705)。将细胞在室温避光温育30分钟。然后用FACS缓冲液将样品洗涤3次以去除任何未结合抗体。在LSRII流式细胞仪上分析样品。根据SSC和FSC特征向活淋巴细胞施加门控(gate)，确定FITC的MFI。
如图3和图4所示，所有含IL-10的构建体均以剂量依赖方式增加STAT3磷酸化。
实施例5
交叉受体结合CD86以及同时结合CD86和IL10
采用表达CD86的人B类淋巴母细胞细胞系(WIL2-S)检测CD86结合，采用在表面表达CD86的CHO细胞系(HuCD86-2A2细胞)以及与鼠IgG Fc连接的可溶性IL10受体1(IL10R1)融合蛋白或抗IL10抗体以检测发现于交叉受体分子上的CD86拮抗物和IL10激动剂的同时结合。简言之，将WIL2-S或HuCD86-2A2细胞与浓度为饱和到背景水平的包含CD86拮抗物的测试分子温育。向HuCD86-2A2细胞再加入IL10R1-muIg融合蛋白或鼠抗IL10抗体，以与已通过CD86与细胞表面结合的测试分子形成复合物。温育后，洗涤细胞并用荧光团(R-藻红蛋白)标记具有交叉受体分子Fc部分、IL10R-Ig融合蛋白或抗IL10抗体特异性的F(Ab’)2抗体。然后使标记细胞通过流式细胞仪，通过对各样品的中值荧光强度作图来分析数据。
如图5所示，通过包含抗CD86结合域(例如，来自杂交瘤抗体3D1和FUN1)的交叉受体分子与WIL2-S细胞上的CD86结合显示的亲和力高于含CTLA4胞外域的交叉受体分子的结合。更具体而言，含抗CD86 3D1的交叉受体3D1::IL10(SEQ ID NO:189)与CD86的亲和力略高于含抗CD86FUN1的交叉受体FUN1::IL10(SEQ ID NO:187)，而CTLA4-Ig(SEQ ID NO:11)和含CTLA4的交叉受体(SEQ ID NO:173)对CD86的亲和力远低于来自杂交瘤抗体3D1和FUN1的抗CD86结合域。
如图6所示，包含CD86拮抗物和IL10激动剂的交叉受体分子可同时结合HuCD86-2A2细胞(内部开发的在细胞表面表达CD86的CHO细胞)上的CD86和可溶性IL10R1。此外，图6显示IL10变体对IL10R1具有不同的结合亲和力。例如，交叉受体分子CTLA4::单IL10(SEQID NO:181)和(CTLA4::IL10)-75(SEQ ID NO:173)具有类似的IL10R1亲和力，但包含病毒突变IL10形式的交叉受体CTLA4::IL10I87A(SEQ ID NO:191)和CTLA4::IL10I87S(SEQ IDNO:193)显示低得多的IL10R1亲和力。
在另一个实验中，测试不同形式的人源化FUN1SMIP的CD86结合。采用HuCD86-2A2细胞检测用6种不同形式的人源化FUN1SMIP瞬时转染的HEK293细胞的上清液的CD86结合。图7显示FUN1-21(SEQ ID NO:227)具有最佳的CD86结合亲和力，之后是FUN1-22(SEQ IDNO:233)，然后是FUN1-11(SEQ ID NO:225)；剩余的人源化FUN1SMIP蛋白(FUN1-12,SEQ IDNO:229；FUN1-31,SEQ ID NO:231；FUN1-32,SEQ ID NO:235)未显示可检测的结合。
实施例6
改变交叉受体蛋白中的结合域接头长度
检测CTLA4::IL10分子的接头稳定性。在CHOK1SV细胞中稳定转染所有接头变体，在37℃温育稳定批量群，并在第3天转移到34℃。通过蛋白A柱和之后的第二步骤SEC柱纯化蛋白质。进行ELISA试验以测定IL10与IL10R1-mIg融合蛋白的结合。图8中的结果显示，由于接头不稳定，交叉受体(CTLA4::IL10)-65(SEQ ID NO:9)与IL10的结合降低，但(CTLA4::IL10)-68(SEQ ID NO:171)、(CTLA4::IL10)-69(SEQ ID NO:302)和(CTLA4::IL10)-75(SEQID NO:173)在这些培养条件下具有稳定性。
还通过ELISA测试了CTLA4::IL10交叉受体蛋白中较短接头变体与IL10R1的结合。瞬时转染较短接头变体，用蛋白A柱纯化蛋白质。进行ELISA试验以测定IL10与IL10R1-mIg融合蛋白的结合。图9中的结果显示，较短接头变体(CTLA4::IL10)-77(SEQ ID NO:175)、Q0033(CTLA4::IL10)-78(SEQ ID NO:177)和(CTLA4::IL10)-79(SEQ ID NO:179)与较长接头(例如，相比(CTLA4::IL10)-68；SEQ ID NO:171)的后端IL10结合亲和力一样良好。
实施例7
交叉受体蛋白的血清稳定性和药代动力学
测试(CTLA4::IL10)-75(SEQ ID NO:173)的血清稳定性。在该实验中，用小鼠血清将SEQ ID NO:173的纯化蛋白在37℃处理24小时，72小时和冻融(F/T)，在试验时间(T0)掺加。测试所有稳定性样品与CD80的结合(采用表达CD80的1F6CHO细胞)以及其与CD80和抗IL10抗体与交叉受体上的IL10的同时结合。结果显示SEQ ID NO:173非常稳定，在与测试浓度(约0.01nM到约10nM)下与小鼠血清温育后保持抗IL10结合。
用雌性Balb/c小鼠进行(CTLA4::IL10)-68(SEQ ID NO:171)和(CTLA4::IL10)-75(SEQ ID NO:173)的药代动力学(PK)研究，每个时间点采用3只小鼠。以200μg/小鼠对小鼠进行静脉内注射。在处理后15分钟、2小时、6小时、24小时、48小时、96小时、7天和14天收集小鼠血清。测试样品的CD80结合(CTLA4鉴定)和与CD80和抗IL10抗体的同时结合(IL10鉴定)。结果总结于以下表4中。
表4PK研究小结
实施例8
交叉受体与CD80、CD86和IL10R1的结合
通过ELISA检测CD80和IL10R1的结合，并/或采用表达CD86的CHO细胞和IL10R1-Ig或抗IL10检测CD86和IL10R1的结合。通过ELISA采用生物素标记的小鼠抗体检测与小鼠CD80和CD86的结合。检测的分子包括对照CTLA4-Ig(SEQ ID NO:11)融合蛋白和以下测试交叉受体分子：(CTLA4::IL10)-65(SEQ ID NO:9)、(CTLA4::IL10)-68(SEQ ID NO:171)、(CTLA4::IL10)-69(SEQ ID NO:302)、(CTLA4::PDL2)-65(SEQ ID NO:336)，基本如实施例3和5所述。
如图10和图11所示，通过ELISA检测，(CTLA4::IL10)-65、(CTLA4::IL10)-68和(CTLA4::IL10)-69交叉受体分子全部结合细胞上的CD80和CD86。图12中的结果显示，CTLA4::IL10交叉受体分子可与huIL10R1相互作用。此外，如图13所示，通过ELISA检测，CTLA4::IL10交叉受体可使BD1(氨基末端结合域)和BD2(羧基末端结合域)同时与CD80和IL10R1发生相互作用。此外，图14和图15显示，具有人分子特异性的(CTLA4::IL10)-65、(CTLA4::IL10)-68和(CTLA4::IL10)-69交叉受体分子能够与小鼠CD80和小鼠CD86发生交叉反应。
实施例9
通过BIAcoreTM检测交叉受体与CD80的结合
检测以下物质的CD80结合活性：阿巴西普(B-M施贵宝公司(Bristol-Myers Squibb))，与贝拉西普类似的包含L104E A29Y突变的CTLA4-Fc融合物(SEQ ID NO:217)，各自具有CTLA4胞外域和IL10结构域的3种CTLA4::IL10交叉受体接头变体(CTLA4::IL10)-65(SEQ ID NO:9)、(CTLA4::IL10)-68(SEQ ID NO:171)和(CTLA4::IL10)-75(SEQID NO:173)，包含具有L104E A29Y突变的CTLA4胞外域和IL10结构域的交叉受体(SEQ IDNO:219)，以及包含CTLA4胞外域和PD-L1结构域的交叉受体(SEQ ID NO:13)，基本如本文所述。之前已描述了通过BIAcore检测CTLA4-Fc与CD80/86的结合(Greene等(1996)J.Biol.Chem.271:26762-26771；van der Merwe等(1997)J.Exp.Med.185:393-403；Collins等(2002)Immunity 17:201-210)。CTLA4与CD80结合具有中等亲和力(Kd＝～200nM)，特征是快速结合速率(4-8×105M-1秒-1)和中等解离速率(0.090秒-1)。二聚体CTLA4-Fc与CD80的结合是双相的，已报道其具有两种解离速率(0.004、0.086秒-1)。已报道CTLA4-Fc上的L104E A29Y突变使CD80亲和力增大到比野生型CTLA4-Fc大两倍(Larsen等(2005)Am.J.Transplant.5:443-453)，其主要降低了初始解离速率(报道为0.00108对0.00221秒-1)。
采用HBS-EP+(GE医疗保健公司)作为运行缓冲液在BIAcoreTM T100SPR(厄普萨拉的法玛西亚生物技术公司)上进行表面等离振子共振(SPR)测定。采用标准胺偶联化学(GE医疗保健公司的Biacore胺偶联试剂盒)将CD80-mIgG(25μg/mL溶于10mM醋酸钠，pH 4.0；安赛尔公司)直接固定到CM5芯片上，最终固定水平为317、973和1678Ru(共振单位)。以10μl/分钟的流速、5nM-1μM的系列浓度注射含CTLA4的构建体，持续150秒。监测解离600秒，通过注射pH 4.0的50mM柠檬酸钠、500mM氯化钠60秒，来再生表面。与表面的结合相互作用在至少75个再生循环中具有稳定性。采用T100的Bia评价软件(版本2.0.1，GE医疗保健公司)来分析数据。
CTLA4构建体与固定化CD80的结合动力学无法用1:1朗缪尔结合模型拟合，但可以高精确度拟合至二价分析物结合模型。通过增加注射时间长度延长解离阶段不改变计算的动力学参数。可通过使观察到的饱和反应拟合至稳态平衡模型来以高精确度计算各构建体的平衡解离常数(KD)。所有CD80结合分子的结果总结于以下表5中。
确定的交叉受体的平衡亲和力与阿巴西普的平衡亲和力和CTLA4-Fc的报道亲和力(200nM；Greene等1996,同上)类似。CTLA4::PDL1交叉受体的结合动力学不同于阿巴西普或CTLA4::IL10交叉受体，虽然结合/解离速率补偿产生类似的亲和力。这可能是由于PD-L1结合CD80的亲和力弱于CTLA4(KD为2.5μM)。与先前的研究类似，含L104E A29Y突变的CTLA4变体(SEQ ID NO：217和219)具有较高的CD80亲和力，其中初始解离速率改善约两倍(相比阿巴西普的0.006秒-1，SEQ ID NO:217为0.00373秒-1)。
实施例10
通过BIAcoreTM检测交叉受体与CD86的结合
检测以下物质的CD86结合活性：阿巴西普，与贝拉西普类似的包含L104E A29Y突变的CTLA4-Fc融合物(SEQ ID NO:217)，包含CTLA4胞外域和IL10结构域的交叉受体(SEQID NO:9)，包含具有L104E A29Y突变的CTLA4胞外域和IL10结构域的交叉受体(SEQ ID NO:219)，以及包含来自3D1和FUN1抗CD86抗体的抗体可变结构域的不同构建体(SMIP、PIMS和交叉受体)，基本如本文所述。CTLA4与CD86的结合具有低亲和力(Kd＝～2.2μM)，其特征是快速结合速率(2-13×105M-1秒-1)和中等解离速率(0.42秒-1)。已报道CTLA4-Fc上的L104EA29Y突变使CD86亲和力增大到比野生型CTLA4-Fc大4倍(Larsen等(2005)Am.J.Transplant.5:443-453)，其主要降低了初始解离速率(报道为0.00206对0.00816秒-1)。很明显，之前未公开3D1或FUN1抗体的动力学或平衡亲和力数据，因此确定其相对亲和力。
基于CTLA4胞外域的较低亲和力CD86结合域。采用HBS-EP+(GE医疗保健公司)作为运行缓冲液在BIAcoreTM T100SPR(厄普萨拉的法玛西亚生物技术公司)上进行SPR测定。采用标准胺偶联化学(GE医疗保健公司的Biacore胺偶联试剂盒)将CD86-mIgG(25μg/mL溶于10mM醋酸钠，pH 4.0；安赛尔公司)直接固定到CM5芯片上，最终固定水平为37、373和903Ru。以10μl/分钟的流速、4nM-10μM的系列浓度注射含CTLA4的构建体150秒。监测解离600秒，通过注射pH 5.0的50mM柠檬酸钠、500mM氯化钠60秒(野生型CTLA4)或注射pH 1.7的10mM甘氨酸(L104E A29Y CTLA4)，来再生表面。与表面的结合相互作用和固定化水平在至少100个再生循环中具有稳定性。采用T100的Bia评价软件(版本2.0.1，GE医疗保健公司)来分析数据。由于CTLA4的CD86亲和力低且结合和解离速率非常快(BIAcoreTM T100仪器的检测限处ka的文献值为0.2-1.3×106M-1秒-1，kd的文献值为0.42秒-1)，无法确定阿巴西普与固定化CD86的结合动力学。然而，可通过使观察到的反应拟合至二价分析物模型以合理的精确度确定含L104E A29Y CTLA4结构域的构建物(SEQ ID NO:217，SEQ ID NO:219)与固定化CD86的结合动力学。可通过使观察到的饱和反应拟合至稳态平衡模型来以高精确度计算所有构建体的平衡解离常数(KD)。结果见以下表6所示。
基于3D1和FUN1抗体可变结构域的较高亲和力CD86结合域。如上所示进行SPR测定，以下为例外：采用HBS-P+(GE医疗保健公司)作为运行缓冲液，监测解离1200秒，通过注射pH 1.7的10mM甘氨酸60秒来再生表面。可确定所有情况中的与固定化CD86的结合动力学。然而，可通过使观察到的饱和反应拟合至稳态平衡模型来以高精确度计算各构建体的平衡解离常数(KD)。结果见以下表6所示。
阿巴西普的平衡亲和力(3.2μM)与CTLA4-Fc的报道亲和力(2.2μM；Greene等1996,同上)类似。与先前的研究类似，含L104E A29Y突变的CTLA4变体(SEQ ID NO:217，SEQ IDNO:219)的CD86亲和力高4-5倍(670-772nM)。在N末端含3D1鼠单链抗体片段(scFv)的构建体(3D1SMIP,SEQ ID NO:317；3D1::IL10,SEQ ID NO:189)的亲和力(11.7,26.5nM)高于在C末端含3D1scFv的相应构建体(3D1PIMS,SEQ ID NO:319；IL10::3D1,SEQ ID NO:185)；检测结合动力学，显示这是由较高的初始结合速率和较低的初始解离速率产生的，但在所有情况中，它们的CD86亲和力均比阿巴西普至少高100倍。就FUN1抗体而言，连同含两个人源化单链FUN1抗体片段的SMIP蛋白(SEQ ID NO:225和227)检测母体鼠单克隆抗体；两种SMIP的后者(SEQ ID NO:227)显示与母体FUN1单克隆抗体类似的结合动力学和CD86总亲和力(分别为26.9、36nM)，这仍显著高于阿巴西普。在羧基末端含人源化FUN1抗体片段的交叉受体或PIMS分子(IL10::FUN1-21,SEQ ID NO:254；(IL10I87A::FUN1-21)-75,SEQ ID NO:258；(单IL10-A2铰链::FUN1-21)-75,(SEQ ID NO:276)；FUN1-21PIMS,SEQ ID NO:402)的亲和力低于在氨基末端含母体抗体序列的交叉受体(FUN1::IL10,SEQ ID NO:187)或SMIP蛋白上的相同FUN1-21结合序列(SEQ ID NO:227)。
实施例11
通过BIAcoreTM检测交叉受体与鼠CD86的结合
检测以下物质的鼠CD86结合活性：阿巴西普，与贝拉西普类似的包含L104E A29Y突变的CTLA4-Fc融合物(SEQ ID NO:217)，包含不同BD2接头但相同CTLA4和IL10结构域的两种交叉受体(SEQ ID NO:171和173)，包含具有L104E A29Y突变的CTLA4胞外域和IL10结构域的交叉受体(SEQ ID NO:219)，鼠CTLA4与人Fc结构域的融合物(SEQ ID NO:404)，以及包含来自大鼠GL1抗鼠CD86抗体的抗体可变区的不同构建体，包括含GL1抗体片段和人IL10结构域的两种交叉受体(GL1::IL10,SEQ ID NO:252；和IL10::GL1,SEQ ID NO:256)，基本如本文所述。已知人CTLA4与鼠CD86发生交叉反应，但就我们目前所知，之前并未描述动力学或亲和力测定。类似地，已将大鼠GL1抗体描述为具有鼠CD86特异性，但并未报道动力学或亲和力测定。
采用HBS-EP+(GE医疗保健公司)作为运行缓冲液在BIAcoreTM T100SPR(厄普萨拉的法玛西亚生物技术公司)上进行SPR测定。采用标准胺偶联化学(GE医疗保健公司的Biacore胺偶联试剂盒)将鼠CD86-mIgG(25μg/mL溶于10mM醋酸钠，pH 4.0；R&D系统公司)直接固定到CM5芯片上，最终固定水平为150、493和746Ru。以10μl/分钟的流速、4nM-8μM的系列浓度注射含CTLA4的构建体150秒。监测解离600秒(CTLA4构建体)或1200秒(GL1构建体)，通过注射pH 1.7的10mM甘氨酸60秒来再生表面。与表面的结合相互作用和固定化水平在至少100个再生循环中具有稳定性。采用T100的Bia评价软件(版本2.0.1，GE医疗保健公司)分析数据。可确定所有构建体与固定化小鼠CD86的结合动力学，并以高精确度拟合至二价分析物模型(表7)。还可通过使观察到的饱和反应拟合至稳态平衡模型来以高精确度计算各构建体的平衡解离常数(KD)。就CTLA4变体(SEQ ID NO:171、173、217、219和404)而言，所有在3种不同固定化密度下的3个流动室上进行同时平衡拟合产生的结果比在任何一个流动室上进行拟合的结果更精确(称为“多R最大”拟合)，因此列出这些亲和力。结果见以下表7所示。
含人CTLA4和人IL10结构域的交叉受体(SEQ ID NO:171和173)对鼠CD86的亲和力(1.54、1.81μM)略高于人CTLA4-Fc对人CD86的报道亲和力(2.2μM；Greene等1996)。含L104EA29Y突变的人CTLA4变体(SEQ ID NO:217和219)的鼠CD86亲和力(870-920nM)只高2倍，这看来是由有益的较高初始小鼠CD86结合速率和有害的较高初始解离速率联合造成的。鼠CTLA4似乎具有与人CTLA4类似或略低的鼠CD86总亲和力。就GL1抗体而言，连同含单链GL1抗体片段的SMIP(GL1SMIP,SEQ ID NO:239)一起检测母体大鼠单克隆抗体；二者对鼠CD86显示类似的结合动力学和总亲和力(分别为37.7、26.2nM)，这显著高于(约50倍)含人或鼠CTLA-4的构建体。含IL-10和GL1抗体片段的交叉受体(SEQ ID NO:252和256)显示比母体抗体或SMIP低3倍的鼠CD86亲和力，但这看来是由各情况中初始结合速率或解离速率降低引起的。
实施例12
通过BIAcoreTM检测交叉受体与PD1的结合
检测PDL1-Fc(SEQ ID NO:268)和PDL2-Fc(SEQ ID NO:270)，以及含CTLA4胞外域和PDL1(SEQ ID NO:13)或PDL2(SEQ ID NO:336)结构域的交叉受体的PD1结合活性，基本如下所述。通常报道PDL2与PD1结合的亲和力高于PDL1与PD1结合；一项先前的动力学分析(Youngnak等,(2003)Biochem.Biophys.Res.Comm.307,672)显示中等的亲和力(PDL1为112nM，PDL2为37nM)，而以替代形式进行的平衡分析(Butte等,(2007)Immunity 27,111)显示较弱的亲和力(PDL1为770nM，PDL2为590nM)。
采用HBS-P+(GE医疗保健公司)作为运行缓冲液在BIAcoreTM T100SPR(厄普萨拉的法玛西亚生物技术公司)上进行SPR测定。最初采用BirA酶(科罗拉多州奥罗拉的亲和力公司(Avidity,Inc.,Aurora,CO))在pH 8.0 10mM Tris中生物素化含与C末端AviTagTM融合的PD1胞外域的构建体(SEQ ID NO:406)，并将缓冲液交换为PBS。采用标准胺偶联化学(GE医疗保健公司的Biacore胺偶联试剂盒)将中性链亲和素(Neutravidin)(100μg/mL溶于10mM醋酸钠，pH 4.0；伊利诺斯州洛克福德的热科学公司(Thermo Scientific,Rockford,IL))直接固定到CM5芯片上，最终固定水平为191、771和1522Ru，分别用来捕获水平为171、597和1244Ru的生物素化PD1。以30μl/分钟的流速、6nM-2μM的系列浓度注射含PDL1/2的构建体120秒。监测解离120秒，通过注射50mM NaOH、1M NaCl 30秒再生表面。与表面的结合相互作用和固定化水平在至少50个再生循环中具有稳定性。
采用T100的Bia评价软件(版本2.0.1，GE医疗保健公司)分析数据。可确定所有构建体与固定化PD1的结合动力学，并以高精确度拟合至二价分析物模型(SEQ ID NO:268和270)或1:1结合模型(SEQ ID NO:13和336)(表8)。还可通过使观察到的饱和反应拟合至稳态平衡模型来以高精确度计算各构建体的平衡解离常数(KD)。结果见以下表8所示。
表8
*文献值(Youngnak等,(2003)Biochem.Biophys.Res.Comm.307,672)
**从第一位点ka和kd计算的动力学KD
PDL1-Fc(SEQ ID NO:268)和PDL2-Fc(SEQ ID NO:270)显示与文献报道类似的结合动力学和总亲和力。羧基末端融合PDL1或PDL2结构域的含CTLA4的交叉受体(SEQ ID NO:13和336)的PD1亲和力(2.36、0.34μM)显著低于(约10倍)氨基末端PDL1/PDL2融合物(246、42nM)；这主要是由初始解离速率的显著升高(0.165、0.0395对0.0544、0.00724)造成的，表明当结合域位于BD2(羧基末端)位置时可使PDL1/2:PD1复合物失稳。
实施例13
交叉受体融合蛋白阻断人T细胞应答
该实施例显示，本发明的交叉受体融合蛋白可阻断人T细胞应答。采用混合淋巴细胞反应(MLR)测试交叉受体融合蛋白的阻断作用。简言之，采用标准方法从两个供体分离人外周血单核细胞(PBMC)，并使其保持分离。根据先前的研究，将来自一个供体的PBMC指定为“应答物”群，将来自另一个供体的PBMC指定为“刺激物”群。采用标准方法用CFSE标记两个供体的PBMC。为防止细胞分裂，用丝裂霉素C(MMC)处理刺激物PBMC。用无菌蒸馏水(吉布可公司(Gibco)#15230)重建MMC(西格玛公司(Sigma)#M4287-2mg)，浓度为0.5mg/ml。将刺激物PBMC以1x106/ml的浓度重悬在完全培养基(CM)(含10％人B血清、100U/ml青霉素、100μg/ml链霉素、2mM L-谷氨酰胺、NEAA、丙酮酸钠、过滤的CM 0.2μm的RPMI-1640)中，加入MMC达到25μg/ml的终浓度。然后在37℃、5％CO2将刺激物PBMC/MMC混合物温育30分钟，之后用CM将细胞洗涤3次。将应答物和刺激物细胞以4x106/ml的浓度重悬在CM中，向96孔平底组织培养板每孔加入每种细胞群各0.05ml，最终达到2x105个细胞/孔/供体。与细胞同时向板中加入图中所示所有指定浓度的处理(注：抗体和融合蛋白的浓度为摩尔当量)。然后在37℃、5％CO2下，用MLR条件(96孔板PBMC处理设置)温育实验持续时间。在第7-8天收获MLR实验，用荧光标记的CD5抗体(e生物科学公司(e-Bioscience))和CD25抗体(BD生物科学公司)对细胞染色，并在流式细胞仪(LSR II，B-D公司(Becton Dickenson))上运行。采用FlowJo流式细胞术软件(树星公司(TreeStar))分析数据。门控方案如下：分析落入FSC:SSC淋巴细胞门的细胞的CD5表达，分析随后落入CD5+门的细胞的CFSE稀释度和CD25上调。将CD5+、CFSE低和CD25高的细胞视作活化T细胞。
图16、17、21和22显示，含异源结合域和CD86拮抗物的多种不同种类交叉受体融合蛋白能够阻断T细胞对应答物/刺激物MLR条件发生应答。
实施例14
CD86拮抗物交叉受体阻断小鼠T细胞应答
采用解剖刀/尼龙网和RBC裂解法分离来自两个不同小鼠品系C57BL/6(或B6D2F1)和BALB/c的小鼠脾细胞。根据先前的研究，将来自小鼠品系C57BL/6(或B6D2F1)的脾细胞指定为“应答物”群，将来自小鼠品系BALB/c的脾细胞指定为“刺激物”群。如前所述用CFSE标记两个小鼠品系的脾细胞。为防止细胞分裂，用丝裂霉素C(MMC)处理刺激物脾细胞。用无菌蒸馏水(吉布可公司(Gibco)#15230)重建MMC(西格玛公司(Sigma)#M4287-2mg)，浓度为0.5mg/ml。将刺激物脾细胞以5x107/ml的浓度重悬在完全培养基(CM)(含10％FBS、100U/ml青霉素、100μg/ml链霉素、2mM L-谷氨酰胺、NEAA、丙酮酸钠和0.05mM 2-巯基乙醇的RPMI-1640)中，加入MMC达到50μg/ml的终浓度。然后在37℃、5％CO2下将刺激物脾细胞/MMC混合物温育20分钟，之后用CM将细胞洗涤3次。将应答物和刺激物细胞以8x106/ml的浓度重悬在CM中，向96孔平底组织培养板每孔加入每种细胞群各0.05ml，最终达到4x105个细胞/孔/品系。与细胞同时向板中加入图18-20中所示所有指定浓度的处理(注：抗体和融合蛋白的所示浓度为摩尔当量)。然后在37℃、5％CO2下用MLR条件(含脾细胞/处理设置的96孔板)温育实验持续时间。在第4-5天收获MLR实验，用荧光标记的CD5抗体(BD生物科学公司)和CD25抗体(BD生物科学公司)对细胞染色，并在流式细胞仪(LSR II，B-D公司)上运行。采用FlowJo流式细胞术软件(树星公司(TreeStar))分析数据。门控方案如下：分析落入FSC:SSC淋巴细胞门的细胞的CD5表达，分析随后落入CD5+门的细胞的CFSE稀释度和CD25上调。将CD5+、CFSE低和CD25高的细胞视作活化T细胞。
图18-20显示，含异源结合域和CD86拮抗物的多种不同种类交叉受体融合蛋白能够阻断小鼠T细胞对应答物(B6D2F1)/刺激物(BALB/c)MLR条件发生应答。
实施例15
含IL10的交叉受体分子的免疫刺激活性
通过体外细胞增殖试验测试各种交叉受体融合蛋白中IL10的免疫刺激活性。具体而言，如下采用MC/9小鼠肥大肝细胞系：在添加10％FBS和5％大鼠T-STIM(BD#354115)的DMEM或RPMI中培养MC/9细胞系(美国典型培养物保藏中心(American Type CultureCollection)#CRL-8306)。洗涤MC/9细胞，并置于不含大鼠T-STIM的培养基中过夜(在37℃、5％CO2温育，96孔平底板中1x 105个细胞/孔，100μl/孔)。即将各种浓度的IL10蛋白和交叉受体融合蛋白温育24小时，6小时后通过[3H]胸苷掺入评价增殖。
已知IL10的I87变体与野生型IL10相比免疫刺激性较小(Ding等,J.Exp.Med.191:213,2000)。IL10通常形成同型二聚体，其中某一单体分子的氨基末端结构域结合于另一个单体羧基末端结构域。连同具有进一步分隔IL10两个亚结构域而使这些亚结构域形成分子内二聚体的较短接头(gggsgg；SEQ ID NO:379)的IL10分子，检测交叉受体形式的IL10I87变体(I87A和I87S)的免疫刺激活性。
图23显示小鼠IL10增强MC/9细胞增殖的能力大于CTLA4::IL10-I87A(SEQ ID NO:191)交叉受，所述CTLA4::IL10-I87A包含IL10的氨基酸87上的单个突变。图24显示人IL10和(CTLA4::IL10)-75(SEQ ID NO:173)增强MC/9细胞增殖的能力大于CTLA4::IL10-I87A(SEQ ID NO:191)或CTLA4::单IL-10(SEQ ID NO:181)。
实施例16
工程改造交叉受体分子以靶向特定细胞类型
该实施例描述工程改造交叉受体融合蛋白以靶向特定细胞类型。这通过工程改造BD1和BD2的亲和力来实现。工程改造对CD86和huIL10R1具有不同亲和力的4种交叉受体分子。表9显示这4种分子的不同亲和力。例如，通过采用CD86结合域(例如，3D1或人源化FUN1)和对huIL10R1具有低亲和力的工程改造IL10分子(I87A)改善CD86亲和力，可采用这样的设置促进靶向感兴趣的特定细胞类型，如APC。
表9
CD86 IL10R1 IL10R1/CD86
BD1 BD2 亲和力(nM) 亲和力(nM) 亲和力比
CLTA4 IL10 2000* 0.1& 0.00005
CTLA4 IL10I87A 2000* 10# 0.005
抗CD86 IL10 40* 0.1& 0.0025
抗CD86 IL10I87A 40* 10# 0.25
*获自CTLA4、3D1和人源化FUN1结合CD86的内部测定的近似平衡亲和力
&基于Tan等(J.Biol.Chem.268:21053,1993)的近似亲和力
#基于Ding等(J.Exp.Med.191:213,2000)的近似亲和力
实施例17
类风湿性关节炎动物模型体内的交叉受体活性
类风湿性关节炎
利用两种类风湿性关节炎(RA)小鼠模型，即胶原诱导性关节炎(CIA)和葡萄糖-6-磷酸异构酶(G6PI)模型中的至少一种模型检测本文所述的任何交叉受体分子的治疗功效。已显示这些模型均可用于预测某类治疗性药物在RA中的功效(参见Holmdahl(2000)Arthritis Res.2：169；Holmdahl(2006)Immunol.Lett.103:86；Holmdahl(2007)MethodsMol.Med.136:185；McDevitt，H.(2000)Arthritis Res.2:85；Kamradt和Schubert(2005)Arthritis Res.Ther.7:20)。
(a)CIA模型
就其发病机理和免疫基础而言，CIA模型是表征最好的关节炎小鼠模型。此外，它是最广泛采用的RA模型，虽然在预测药物抑制患者疾病的能力方面并不完美，但仍有许多人认为它是研究RA潜在新治疗方法的良好模型(Jirholt等(2001)Arthritis Res.3:87；Van den Berg，W.B.(2002)Curr.Rheumatol.Rep.4:232；Rosloniec(2003)“胶原诱导性关节炎(Collagen-Induced Arthritis)”.刊于《新编免疫学研究方法(Current Protocolsin Immunology)》，Coligan等编，新泽西州霍波肯的约翰韦利森公司(John Wiley&Sons，Inc，Hoboken，NJ))。
在CIA模型中，通过用完全弗氏佐剂(CFA)配制的胶原II(CII)免疫雄性DBA/1小鼠来诱导关节炎。具体而言，在第21天对小鼠皮内/皮下注射CFA配制的CII，在第0天用不完全弗氏佐剂(IFA)配制的CII加强免疫。在用CII/IFA加强免疫后若干天内，小鼠出现关节炎的临床迹象。一小组用CII/IFA免疫的小鼠(0％-10％)未经加强免疫时，在第0天或其左右出现关节炎迹象，从本实验中排除这些动物。在一些CIA实验中，省去加强免疫，而是在CII/CFA免疫后21天(即第一次处理之日是第0天)开始用交叉受体或对照处理小鼠。
在预防和/或治疗方案中用交叉受体、载体(PBS)或阴性或阳性对照处理小鼠。预防性处理从第0天开始，一直持续到对照(未处理)小鼠的疾病峰值后。治疗性处理从大部分小鼠出现轻微关节炎迹象时开始。将已显示对CIA和G6PI诱导的关节炎模型均有良好功效的恩利用作阳性对照。在每个实验中采集的数据包括关节炎的临床评分和累积发病率。用0-4分的标准来评价CIA模型的临床关节炎迹象，如以下表10所示。
表10
(b)G6PI模型
在G6PI模型中，用佐剂配制的G6PI免疫DBA/1小鼠以诱导关节炎(Kamradt和Schubert(2005)Arthritis Res.Ther.7:20；Schubert等(2004)J.Immunol.172:4503；Bockermann，R.等(2005)Arthritis Res.Ther.7:R1316；Iwanami等(2008)ArthritisRheum.58:754；Matsumoto等(2008)Arthritis Res.Ther.10:R66)。G6PI是体内基本所有细胞中存在的酶，但尚不清楚为何免疫诱导特定关节的疾病。多种试剂，如CTLA4-Ig、TNF拮抗物(例如)和抗IL6受体单克隆抗体均显示在G6PI模型中抑制关节炎的发生。
用完全弗氏佐剂(CFA)配制的G6PI免疫雄性DBA/1小鼠，以诱导关节炎。具体而言，在第0天对小鼠皮内/皮下注射CFA配制的G6PI，在免疫数天内小鼠出现临床关节炎迹象。如上述CIA模型的情况那样，在预防和/或治疗方案用交叉受体、载体(PBS)或阴性或阳性对照处理小鼠。预防性处理从第0天开始，一直持续到对照小鼠的疾病峰值后。治疗性处理从大部分小鼠出现轻微关节炎迹象时开始。将已显示对CIA和G6PI诱导的关节炎模型均有良好功效的用作阳性对照。在每个实验中采集的数据包括关节炎的临床评分和累积发病率。用类似于CIA模型所用的标准来评价G6PI模型的临床关节炎迹象。
尽管已经结合本文所述具体实施方式对本发明进行了描述，但很明显，本领域技术人员不难进行多种替代、改良和变化。因此，以上所列的本发明实施方式应为说明性，而非限制性。可在不背离所附权利要求书定义的本发明精神和范围的情况下进行各种改变。本说明书提到的和/或申请数据单中列出的所有专利、专利申请公开、专利申请和非专利出版物均通过引用全文纳入本文。
序列表
<110> P.A.汤普森 (Thompson, Peter A.)
P.R.包姆(Baum, Peter R.)
P.丹(Tan, Phillip)
J.W.布兰肯什普(Blankenship, John W.)
S.纳塔拉加(Natarajan, Sateesh)
<120> CD86拮抗物多靶点结合蛋白
<130> 910180.421PC
<140> PCT
<141> 2009-10-02
<160> 422
<170> FastSEQ Windows 4.0版
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Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu
145 150 155 160
Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile
165 170 175
Arg Asn
<210> 8
<211> 1700
<212> DNA
<213> 人工序列
<220>
<223> 编码具有CTLA4胞外域和IL10结构域的融合蛋白的多核苷酸
<400> 8
aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60
gataccaccg gtatgcacgt ggcccagcct gctgtggtac tggccagcag ccgaggcatc 120
gccagctttg tgtgtgagta tgcatctcca ggcaaagcca ctgaggtccg ggtgacagtg 180
cttcggcagg ctgacagcca ggtgactgaa gtctgtgcgg caacctacat gatggggaat 240
gagttgacct tcctagatga ttccatctgc acgggcacct ccagtggaaa tcaagtgaac 300
ctcactatcc aaggactgag ggccatggac acgggactct acatctgcaa ggtggagctc 360
atgtacccac cgccatacta cctgggcata ggcaacggaa cccagattta tgtaattgat 420
ccagaaccgt gcccagattc tgacctcgag cccaaatctt ctgacaaaac tcacacatgc 480
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 540
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 600
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 660
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 720
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 780
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 840
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 900
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 960
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1020
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1080
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1140
cagaggcaca acaattcttc cctgaataca agaactcaga aagcacgtca ttctccgaat 1200
tctagcccag gccagggcac ccagtctgag aacagctgca cccacttccc aggcaacctg 1260
cctaacatgc ttcgagatct ccgagatgcc ttcagcagag tgaagacttt ctttcaaatg 1320
aaggatcagc tggacaactt gttgttaaag gagtccttgc tggaggactt taagggttac 1380
ctgggttgcc aagccttgtc tgagatgatc cagttttacc tggaggaggt gatgccccaa 1440
gctgagaacc aagacccaga catcaaggcg catgtgaact ccctggggga gaacctgaag 1500
accctcaggc tgaggctacg gcgctgtcat cgatttcttc cctgtgaaaa caagagcaag 1560
gccgtggagc aggtgaagaa tgcctttaat aagctccaag agaaaggcat ctacaaagcc 1620
atgagtgagt ttgacatctt catcaactac atagaagcct acatgacaat gaagatacga 1680
aactaatcta gagcggccgc 1700
<210> 9
<211> 537
<212> PRT
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和IL10结构域的融合蛋白
<400> 9
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr
355 360 365
Gln Lys Ala Arg His Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln
370 375 380
Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu
385 390 395 400
Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met
405 410 415
Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp
420 425 430
Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe
435 440 445
Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile
450 455 460
Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu
465 470 475 480
Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys
485 490 495
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly
500 505 510
Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu
515 520 525
Ala Tyr Met Thr Met Lys Ile Arg Asn
530 535
<210> 10
<211> 1158
<212> DNA
<213> 人工序列
<220>
<223> 编码CTLA4-Ig构建体的多核苷酸
<400> 10
aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60
gataccaccg gtatgcacgt ggcccagcct gctgtggtac tggccagcag ccgaggcatc 120
gccagctttg tgtgtgagta tgcatctcca ggcaaagcca ctgaggtccg ggtgacagtg 180
cttcggcagg ctgacagcca ggtgactgaa gtctgtgcgg caacctacat gatggggaat 240
gagttgacct tcctagatga ttccatctgc acgggcacct ccagtggaaa tcaagtgaac 300
ctcactatcc aaggactgag ggccatggac acgggactct acatctgcaa ggtggagctc 360
atgtacccac cgccatacta cctgggcata ggcaacggaa cccagattta tgtaattgat 420
ccagaaccgt gcccagattc tgacctcgag cccaaatctt ctgacaaaac tcacacatgc 480
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 540
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 600
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 660
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 720
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 780
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 840
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 900
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 960
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1020
tacagcaagc tcaccgtgga caagagccgg tggcagcagg ggaacgtctt ctcatgctcc 1080
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1140
taatctagag cggccgcc 1158
<210> 11
<211> 356
<212> PRT
<213> 人工序列
<220>
<223> CTLA4-Ig构建体
<400> 11
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly
355
<210> 12
<211> 1880
<212> DNA
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和PDL1结构域的融合蛋白的核苷酸表达盒
<400> 12
aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60
gataccaccg gtatgcacgt ggcccagcct gctgtggtac tggccagcag ccgaggcatc 120
gccagctttg tgtgtgagta tgcatctcca ggcaaagcca ctgaggtccg ggtgacagtg 180
cttcggcagg ctgacagcca ggtgactgaa gtctgtgcgg caacctacat gatggggaat 240
gagttgacct tcctagatga ttccatctgc acgggcacct ccagtggaaa tcaagtgaac 300
ctcactatcc aaggactgag ggccatggac acgggactct acatctgcaa ggtggagctc 360
atgtacccac cgccatacta cctgggcata ggcaacggaa cccagattta tgtaattgat 420
ccagaaccgt gcccagattc tgacctcgag cccaaatctt ctgacaaaac tcacacatgc 480
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 540
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 600
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 660
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 720
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 780
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 840
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 900
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 960
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1020
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1080
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1140
cagaggcaca acaattcttc cctgaataca agaactcaga aagcacgtca ttctccgaat 1200
tcttttactg tcacggttcc caaggaccta tatgtggtag agtatggtag caatatgaca 1260
attgaatgca aattcccagt agaaaaacaa ttagacctgg ctgcactaat tgtctattgg 1320
gaaatggagg ataagaacat tattcaattt gtgcatggag aggaagacct gaaggttcag 1380
catagtagct acagacagag ggcccggctg ttgaaggacc agctctccct gggaaatgct 1440
gcacttcaga tcacagatgt gaaattgcag gatgcagggg tgtaccgctg catgatcagc 1500
tatggtggtg ccgactacaa gcgaattact gtgaaagtca atgccccata caacaaaatc 1560
aaccaaagaa ttttggttgt ggatccagtc acctctgaac atgaactgac atgtcaggct 1620
gagggctacc ccaaggccga agtcatctgg acaagcagtg accatcaagt cctgagtggt 1680
aagaccacca ccaccaattc caagagagag gagaagcttt tcaatgtgac cagcacactg 1740
agaatcaaca caacaactaa tgagattttc tactgcactt ttaggagatt agatcctgag 1800
gaaaaccata cagctgaatt ggtcatccca gaactacctc tggcacatcc tccaaatgaa 1860
aggtaatcta gagcggccgc 1880
<210> 13
<211> 597
<212> PRT
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和PDL1结构域的融合蛋白
<400> 13
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr
355 360 365
Gln Lys Ala Arg His Ser Pro Asn Ser Phe Thr Val Thr Val Pro Lys
370 375 380
Asp Leu Tyr Val Val Glu Tyr Gly Ser Asn Met Thr Ile Glu Cys Lys
385 390 395 400
Phe Pro Val Glu Lys Gln Leu Asp Leu Ala Ala Leu Ile Val Tyr Trp
405 410 415
Glu Met Glu Asp Lys Asn Ile Ile Gln Phe Val His Gly Glu Glu Asp
420 425 430
Leu Lys Val Gln His Ser Ser Tyr Arg Gln Arg Ala Arg Leu Leu Lys
435 440 445
Asp Gln Leu Ser Leu Gly Asn Ala Ala Leu Gln Ile Thr Asp Val Lys
450 455 460
Leu Gln Asp Ala Gly Val Tyr Arg Cys Met Ile Ser Tyr Gly Gly Ala
465 470 475 480
Asp Tyr Lys Arg Ile Thr Val Lys Val Asn Ala Pro Tyr Asn Lys Ile
485 490 495
Asn Gln Arg Ile Leu Val Val Asp Pro Val Thr Ser Glu His Glu Leu
500 505 510
Thr Cys Gln Ala Glu Gly Tyr Pro Lys Ala Glu Val Ile Trp Thr Ser
515 520 525
Ser Asp His Gln Val Leu Ser Gly Lys Thr Thr Thr Thr Asn Ser Lys
530 535 540
Arg Glu Glu Lys Leu Phe Asn Val Thr Ser Thr Leu Arg Ile Asn Thr
545 550 555 560
Thr Thr Asn Glu Ile Phe Tyr Cys Thr Phe Arg Arg Leu Asp Pro Glu
565 570 575
Glu Asn His Thr Ala Glu Leu Val Ile Pro Glu Leu Pro Leu Ala His
580 585 590
Pro Pro Asn Glu Arg
595
<210> 14
<211> 338
<212> PRT
<213> 智人(Homo sapiens)
<400> 14
Met Val Val Met Ala Pro Arg Thr Leu Phe Leu Leu Leu Ser Gly Ala
1 5 10 15
Leu Thr Leu Thr Glu Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe
20 25 30
Ser Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala
35 40 45
Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ser
50 55 60
Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu Gln Glu Gly
65 70 75 80
Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala His Ala Gln
85 90 95
Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr Asn Gln Ser
100 105 110
Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys Asp Leu Gly
115 120 125
Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala Tyr Asp Gly
130 135 140
Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala
145 150 155 160
Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala Ala Asn Val
165 170 175
Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu
180 185 190
His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg Ala Asp Pro
195 200 205
Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr Glu Ala Thr
210 215 220
Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Ile Leu Thr
225 230 235 240
Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu Leu Val Glu
245 250 255
Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val
260 265 270
Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu
275 280 285
Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Lys Gln Ser Ser Leu Pro
290 295 300
Thr Ile Pro Ile Met Gly Ile Val Ala Gly Leu Val Val Leu Ala Ala
305 310 315 320
Val Val Thr Gly Ala Ala Val Ala Ala Val Leu Trp Arg Lys Lys Ser
325 330 335
Ser Asp
<210> 15
<211> 318
<212> PRT
<213> 智人(Homo sapiens)
<400> 15
Met Val Val Met Ala Pro Arg Thr Leu Phe Leu Leu Leu Ser Gly Ala
1 5 10 15
Leu Thr Leu Thr Glu Thr Trp Gly Ser His Ser Met Arg Tyr Phe Ser
20 25 30
Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala Met
35 40 45
Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ser Ala
50 55 60
Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu Gln Glu Gly Pro
65 70 75 80
Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala His Ala Gln Thr
85 90 95
Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr Asn Gln Ser Glu
100 105 110
Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys Asp Leu Gly Ser
115 120 125
Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys
130 135 140
Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala Asp
145 150 155 160
Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala Ala Asn Val Ala
165 170 175
Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu His
180 185 190
Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg Ala Asp Pro Pro
195 200 205
Lys Thr His Val Thr His His Pro Val Phe Asp Tyr Glu Ala Thr Leu
210 215 220
Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Ile Leu Thr Trp
225 230 235 240
Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu Leu Val Glu Thr
245 250 255
Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val Val
260 265 270
Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu Gly
275 280 285
Leu Pro Glu Pro Leu Met Leu Arg Trp Ser Lys Glu Gly Asp Gly Gly
290 295 300
Ile Met Ser Val Arg Glu Ser Arg Ser Leu Ser Glu Asp Leu
305 310 315
<210> 16
<211> 2105
<212> DNA
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和HLA-G5结构域的融合蛋白的核苷酸表达盒
<400> 16
aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60
gataccaccg gtatgcacgt ggcccagcct gctgtggtac tggccagcag ccgaggcatc 120
gccagctttg tgtgtgagta tgcatctcca ggcaaagcca ctgaggtccg ggtgacagtg 180
cttcggcagg ctgacagcca ggtgactgaa gtctgtgcgg caacctacat gatggggaat 240
gagttgacct tcctagatga ttccatctgc acgggcacct ccagtggaaa tcaagtgaac 300
ctcactatcc aaggactgag ggccatggac acgggactct acatctgcaa ggtggagctc 360
atgtacccac cgccatacta cctgggcata ggcaacggaa cccagattta tgtaattgat 420
ccagaaccgt gcccagattc tgacctcgag cccaaatctt ctgacaaaac tcacacatgc 480
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 540
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 600
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 660
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 720
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 780
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 840
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 900
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 960
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1020
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1080
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1140
cagaggcaca acaattcttc cctgaataca agaactcaga aagcacgtca ttctccgaat 1200
tctggctccc actccatgag gtatttcagc gccgccgtgt cccggcccgg ccgcggggag 1260
ccccgcttca tcgccatggg ctacgtggac gacacgcagt tcgtgcggtt cgacagcgac 1320
tcggcgtgtc cgaggatgga gccgcgggcg ccgtgggtgg agcaggaggg gccggagtat 1380
tgggaagagg agacacggaa caccaaggcc cacgcacaga ctgacagaat gaacctgcag 1440
accctgcgcg gctactacaa ccagagcgag gccagttctc acaccctcca gtggatgatt 1500
ggctgcgacc tggggtccga cggacgcctc ctccgcgggt atgaacagta tgcctacgat 1560
ggcaaggatt acctcaccct gaacgaggac ctgcgctcct ggaccgcagc ggacactgcg 1620
gctcagatct ccaagcgcaa gtgtgaggcg gccaatgtgg ctgaacaaag gagagcctac 1680
ctggagggca cgtgcgtgga gtggctccac agatacctgg agaacgggaa ggagatgctg 1740
cagcgcgcgg acccccccaa gacacacgtg acccaccacc ctgtctttga ctatgaggcc 1800
accctgaggt gctgggccct gggcttctac cctgcggaga tcatactgac ctggcagcgg 1860
gatggggagg accagaccca ggacgtggag ctcgtggaga ccaggcctgc aggggatgga 1920
accttccaga agtgggcagc tgtggtggtg ccttctggag aggagcagag atacacgtgc 1980
catgtgcagc atgaggggct gccggagccc ctcatgctga gatggagtaa ggagggagat 2040
ggaggcatca tgtctgttag ggaaagcagg agcctctctg aagaccttta atctagagcg 2100
gccgc 2105
<210> 17
<211> 672
<212> PRT
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和HLA-G5结构域的融合蛋白
<400> 17
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr
355 360 365
Gln Lys Ala Arg His Ser Pro Asn Ser Gly Ser His Ser Met Arg Tyr
370 375 380
Phe Ser Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile
385 390 395 400
Ala Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp
405 410 415
Ser Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu Gln Glu
420 425 430
Gly Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala His Ala
435 440 445
Gln Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr Asn Gln
450 455 460
Ser Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys Asp Leu
465 470 475 480
Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala Tyr Asp
485 490 495
Gly Lys Asp Tyr Leu Thr Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala
500 505 510
Ala Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala Ala Asn
515 520 525
Val Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp
530 535 540
Leu His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg Ala Asp
545 550 555 560
Pro Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr Glu Ala
565 570 575
Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Ile Leu
580 585 590
Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu Leu Val
595 600 605
Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val
610 615 620
Val Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His
625 630 635 640
Glu Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Ser Lys Glu Gly Asp
645 650 655
Gly Gly Ile Met Ser Val Arg Glu Ser Arg Ser Leu Ser Glu Asp Leu
660 665 670
<210> 18
<211> 728
<212> PRT
<213> 智人(Homo sapiens)
<400> 18
Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu
1 5 10 15
Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln
20 25 30
Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr
35 40 45
Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val
50 55 60
Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu
65 70 75 80
Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys
85 90 95
Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe
100 105 110
Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys
115 120 125
Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys
130 135 140
Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His
145 150 155 160
Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr
165 170 175
Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser
180 185 190
Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu
195 200 205
Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp
210 215 220
His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro
225 230 235 240
His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp
245 250 255
Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr
260 265 270
Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys
275 280 285
Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu
290 295 300
Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile
305 310 315 320
Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu
325 330 335
His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn
340 345 350
Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr
355 360 365
Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp
370 375 380
Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met
385 390 395 400
Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp
405 410 415
Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala
420 425 430
Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His
435 440 445
Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys
450 455 460
Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu
465 470 475 480
Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val
485 490 495
Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg
500 505 510
Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp
515 520 525
Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr
530 535 540
Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys
545 550 555 560
Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly
565 570 575
Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp
580 585 590
Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu
595 600 605
Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn
610 615 620
Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu
625 630 635 640
Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu
645 650 655
Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp
660 665 670
Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu
675 680 685
Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly
690 695 700
Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile
705 710 715 720
Leu Thr Tyr Lys Val Pro Gln Ser
725
<210> 19
<211> 290
<212> PRT
<213> 智人(Homo sapiens)
<400> 19
Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu
1 5 10 15
Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln
20 25 30
Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr
35 40 45
Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val
50 55 60
Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu
65 70 75 80
Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys
85 90 95
Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe
100 105 110
Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys
115 120 125
Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys
130 135 140
Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His
145 150 155 160
Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr
165 170 175
Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser
180 185 190
Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu
195 200 205
Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp
210 215 220
His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro
225 230 235 240
His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp
245 250 255
Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr
260 265 270
Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys
275 280 285
Glu Thr
290
<210> 20
<211> 723
<212> PRT
<213> 智人(Homo sapiens)
<400> 20
Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu
1 5 10 15
Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln
20 25 30
Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr
35 40 45
Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val
50 55 60
Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu
65 70 75 80
Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys
85 90 95
Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe
100 105 110
Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys
115 120 125
Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys
130 135 140
Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His
145 150 155 160
Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg
165 170 175
Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg
180 185 190
Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr
195 200 205
Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly
210 215 220
Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys Phe
225 230 235 240
Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg
245 250 255
Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro His
260 265 270
Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys Ala Asp Asn Thr Met
275 280 285
Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu Cys Ile Gln Gly Gln
290 295 300
Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile Pro
305 310 315 320
Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met Thr Pro
325 330 335
Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro
340 345 350
Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg
355 360 365
Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp Met Ser His Gly Gln
370 375 380
Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser Gln
385 390 395 400
Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu Asp
405 410 415
Leu His Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Glu
420 425 430
Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr
435 440 445
Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys
450 455 460
Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile
465 470 475 480
Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro Thr
485 490 495
Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn Lys His
500 505 510
Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val Leu Thr Ala Arg
515 520 525
Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly
530 535 540
Ile His Asp Val His Gly Arg Gly Asp Glu Lys Cys Lys Gln Val Leu
545 550 555 560
Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu
565 570 575
Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp Phe Val Ser Thr Ile
580 585 590
Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu Lys Thr Ser Cys Ser
595 600 605
Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn Tyr Asp Gly Leu Leu
610 615 620
Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu Lys Cys Ser Gln His
625 630 635 640
His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu Ile Cys Ala Gly Ala
645 650 655
Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu
660 665 670
Val Cys Glu Gln His Lys Met Arg Met Val Leu Gly Val Ile Val Pro
675 680 685
Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly Ile Phe Val Arg Val
690 695 700
Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile Leu Thr Tyr Lys Val
705 710 715 720
Pro Gln Ser
<210> 21
<211> 285
<212> PRT
<213> 智人(Homo sapiens)
<400> 21
Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu
1 5 10 15
Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln
20 25 30
Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr
35 40 45
Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val
50 55 60
Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu
65 70 75 80
Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys
85 90 95
Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe
100 105 110
Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys
115 120 125
Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys
130 135 140
Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His
145 150 155 160
Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg
165 170 175
Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg
180 185 190
Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr
195 200 205
Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly
210 215 220
Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys Phe
225 230 235 240
Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg
245 250 255
Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro His
260 265 270
Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys Glu Thr
275 280 285
<210> 22
<211> 210
<212> PRT
<213> 智人(Homo sapiens)
<400> 22
Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu
1 5 10 15
Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln
20 25 30
Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr
35 40 45
Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val
50 55 60
Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu
65 70 75 80
Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys
85 90 95
Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe
100 105 110
Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys
115 120 125
Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys
130 135 140
Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His
145 150 155 160
Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr
165 170 175
Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser
180 185 190
Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu
195 200 205
Gly Lys
210
<210> 23
<211> 3794
<212> DNA
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和HGF结构域的融合蛋白的核苷酸表达盒
<400> 23
aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60
gataccaccg gtatgcacgt ggcccagcct gctgtggtac tggccagcag ccgaggcatc 120
gccagctttg tgtgtgagta tgcatctcca ggcaaagcca ctgaggtccg ggtgacagtg 180
cttcggcagg ctgacagcca ggtgactgaa gtctgtgcgg caacctacat gatggggaat 240
gagttgacct tcctagatga ttccatctgc acgggcacct ccagtggaaa tcaagtgaac 300
ctcactatcc aaggactgag ggccatggac acgggactct acatctgcaa ggtggagctc 360
atgtacccac cgccatacta cctgggcata ggcaacggaa cccagattta tgtaattgat 420
ccagaaccgt gcccagattc tgacctcgag cccaaatctt ctgacaaaac tcacacatgc 480
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 540
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 600
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 660
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 720
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 780
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 840
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 900
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 960
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1020
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1080
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1140
cagaggcaca acaattcttc cctgaataca agaactcaga aagcacgtca ttctccgaat 1200
tctagcccag gccagggcac ccagtctgag aacagctgca cccacttccc aggcaacctg 1260
cctaacatgc ttcgagatct ccgagatgcc ttcagcagag tgaagacttt ctttcaaatg 1320
aaggatcagc tggacaactt gttgttaaag gagtccttgc tggaggactt taagggttac 1380
ctgggttgcc aagccttgtc tgagatgatc cagttttacc tggaggaggt gatgccccaa 1440
gctgagaacc aagacccaga catcaaggcg catgtgaact ccctggggga gaacctgaag 1500
accctcaggc tgaggctacg gcgctgtcat cgatttcttc cctgtgaaaa caagagcaag 1560
gccgtggagc aggtgaagaa tgcctttaat aagctccaag agaaaggcat ctacaaagcc 1620
atgagtgagt ttgacatctt catcaactac atagaagcct acatgacaat gaagatacga 1680
aactaatcta gagcggccgc caaaggaaaa gaagaaatac aattcatgaa ttcaaaaaat 1740
cagcaaagac taccctaatc aaaatagatc cagcactgaa gataaaaacc aaaaaagtga 1800
atactgcaga ccaatgtgct aatagatgta ctaggaataa aggacttcca ttcacttgca 1860
aggcttttgt ttttgataaa gcaagaaaac aatgcctctg gttccccttc aatagcatgt 1920
caagtggagt gaaaaaagaa tttggccatg aatttgacct ctatgaaaac aaagactaca 1980
ttagaaactg catcattggt aaaggacgca gctacaaggg aacagtatct atcactaaga 2040
gtggcatcaa atgtcagccc tggagttcca tgataccaca cgaacacagc tttttgcctt 2100
cgagctatcg gggtaaagac ctacaggaaa actactgtcg aaatcctcga ggggaagaag 2160
ggggaccctg gtgtttcaca agcaatccag aggtacgcta cgaagtctgt gacattcctc 2220
agtgttcaga agttgaatgc atgacctgca atggggagag ttatcgaggt ctcatggatc 2280
atacagaatc aggcaagatt tgtcagcgct gggatcatca gacaccacac cggcacaaat 2340
tcttgcctga aagatatccc gacaagggct ttgatgataa ttattgccgc aatcccgatg 2400
gccagccgag gccatggtgc tatactcttg accctcacac ccgctgggag tactgtgcaa 2460
ttaaaacatg cgctgacaat actatgaatg acactgatgt tcctttggaa acaactgaat 2520
gcatccaagg tcaaggagaa ggctacaggg gcactgtcaa taccatttgg aatggaattc 2580
catgtcagcg ttgggattct cagtatcctc acgagcatga catgactcct gaaaatttca 2640
agtgcaagga cctacgagaa aattactgcc gaaatccaga tgggtctgaa tcaccctggt 2700
gttttaccac tgatccaaac atccgagttg gctactgctc ccaaattcca aactgtgata 2760
tgtcacatgg acaagattgt tatcgtggga atggcaaaaa ttatatgggc aacttatccc 2820
aaacaagatc tggactaaca tgttcaatgt gggacaagaa catggaagac ttacatcgtc 2880
atatcttctg ggaaccagat gcaagtaagc tgaatgagaa ttactgccga aatccagatg 2940
atgatgctca tggaccctgg tgctacacgg gaaatccact cattccttgg gattattgcc 3000
ctatttctcg ttgtgaaggt gataccacac ctacaatagt caatttagac catcccgtaa 3060
tatcttgtgc caaaacgaaa caattgcgag ttgtaaatgg gattccaaca cgaacaaaca 3120
taggatggat ggttagtttg agatacagaa ataaacatat ctgcggagga tcattgataa 3180
aggagagttg ggttcttact gcacgacagt gtttcccttc tcgagacttg aaagattatg 3240
aagcttggct tggaattcat gatgtccacg gaagaggaga tgagaaatgc aaacaggttc 3300
tcaatgtttc ccagctggta tatggccctg aaggatcaga tctggtttta atgaagcttg 3360
ccaggcctgc tgtcctggat gattttgtta gtacgattga tttacctaat tatggatgca 3420
caattcctga aaagaccagt tgcagtgttt atggctgggg ctacactgga ttgatcaact 3480
atgatggcct attacgagtg gcacatctct atataatggg aaatgagaaa tgcagccagc 3540
atcatcgagg gaaggtgact ctgaatgagt ctgaaatatg tgctggggct gaaaagattg 3600
gatcaggacc atgtgagggg gattatggtg gcccacttgt ttgtgagcaa cataaaatga 3660
gaatggttct tggtgtcatt gttcctggtc gtggatgtgc cattccaaat cgtcctggta 3720
tttttgtccg agtagcatat tatgcaaaat ggatacacaa aattatttta acatataagg 3780
taccacagtc atag 3794
<210> 24
<211> 1074
<212> PRT
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和HGF结构域的融合蛋白
<400> 24
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr
355 360 365
Gln Lys Ala Arg His Ser Pro Asn Ser Gln Arg Lys Arg Arg Asn Thr
370 375 380
Ile His Glu Phe Lys Lys Ser Ala Lys Thr Thr Leu Ile Lys Ile Asp
385 390 395 400
Pro Ala Leu Lys Ile Lys Thr Lys Lys Val Asn Thr Ala Asp Gln Cys
405 410 415
Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu Pro Phe Thr Cys Lys Ala
420 425 430
Phe Val Phe Asp Lys Ala Arg Lys Gln Cys Leu Trp Phe Pro Phe Asn
435 440 445
Ser Met Ser Ser Gly Val Lys Lys Glu Phe Gly His Glu Phe Asp Leu
450 455 460
Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys Ile Ile Gly Lys Gly Arg
465 470 475 480
Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln
485 490 495
Pro Trp Ser Ser Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser
500 505 510
Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly
515 520 525
Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr
530 535 540
Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr Cys
545 550 555 560
Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly Lys
565 570 575
Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys Phe Leu
580 585 590
Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg Asn
595 600 605
Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro His Thr
610 615 620
Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys Ala Asp Asn Thr Met Asn
625 630 635 640
Asp Thr Asp Val Pro Leu Glu Thr Thr Glu Cys Ile Gln Gly Gln Gly
645 650 655
Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile Pro Cys
660 665 670
Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met Thr Pro Glu
675 680 685
Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro Asp
690 695 700
Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg Val
705 710 715 720
Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp Met Ser His Gly Gln Asp
725 730 735
Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser Gln Thr
740 745 750
Arg Ser Gly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu Asp Leu
755 760 765
His Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Glu Asn
770 775 780
Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr Thr
785 790 795 800
Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys Glu
805 810 815
Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile Ser
820 825 830
Cys Ala Lys Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro Thr Arg
835 840 845
Thr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn Lys His Ile
850 855 860
Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val Leu Thr Ala Arg Gln
865 870 875 880
Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly Ile
885 890 895
His Asp Val His Gly Arg Gly Asp Glu Lys Cys Lys Gln Val Leu Asn
900 905 910
Val Ser Gln Leu Val Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu Met
915 920 925
Lys Leu Ala Arg Pro Ala Val Leu Asp Asp Phe Val Ser Thr Ile Asp
930 935 940
Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu Lys Thr Ser Cys Ser Val
945 950 955 960
Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn Tyr Asp Gly Leu Leu Arg
965 970 975
Val Ala His Leu Tyr Ile Met Gly Asn Glu Lys Cys Ser Gln His His
980 985 990
Arg Gly Lys Val Thr Leu Asn Glu Ser Glu Ile Cys Ala Gly Ala Glu
995 1000 1005
Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu Val
1010 1015 1020
Cys Glu Gln His Lys Met Arg Met Val Leu Gly Val Ile Val Pro Gly
1025 1030 1035 1040
Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly Ile Phe Val Arg Val Ala
1045 1050 1055
Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile Leu Thr Tyr Lys Val Pro
1060 1065 1070
Gln Ser
<210> 25
<211> 229
<212> PRT
<213> 智人(Homo sapiens)
<400> 25
Met Thr Pro Gln Leu Leu Leu Ala Leu Val Leu Trp Ala Ser Cys Pro
1 5 10 15
Pro Cys Ser Gly Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu Pro Arg
20 25 30
Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys Ser Trp
35 40 45
Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro Val Ser Phe Ile Ala
50 55 60
Thr Tyr Arg Leu Gly Met Ala Ala Arg Gly His Ser Trp Pro Cys Leu
65 70 75 80
Gln Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile Thr Asp Val Gln Leu
85 90 95
Phe Ser Met Ala Pro Tyr Val Leu Asn Val Thr Ala Val His Pro Trp
100 105 110
Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr Glu His Ile Ile Lys
115 120 125
Pro Asp Pro Pro Glu Gly Val Arg Leu Ser Pro Leu Ala Glu Arg Gln
130 135 140
Leu Gln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro Glu Ile
145 150 155 160
Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln Gly Ala Ala Arg
165 170 175
Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser Phe Ile Leu Arg Ala
180 185 190
Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val Ala Ala Gln Asp Leu
195 200 205
Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser Leu Pro Ala Thr Ala Thr
210 215 220
Met Ser Leu Gly Lys
225
<210> 26
<211> 253
<212> PRT
<213> 智人(Homo sapiens)
<400> 26
Met Trp Pro Pro Gly Ser Ala Ser Gln Pro Pro Pro Ser Pro Ala Ala
1 5 10 15
Ala Thr Gly Leu His Pro Ala Ala Arg Pro Val Ser Leu Gln Cys Arg
20 25 30
Leu Ser Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val
35 40 45
Leu Leu Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro
50 55 60
Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
65 70 75 80
Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr
85 90 95
Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys
100 105 110
Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu
115 120 125
Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys
130 135 140
Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser
145 150 155 160
Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn
165 170 175
Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn
180 185 190
Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
195 200 205
Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys
210 215 220
Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala
225 230 235 240
Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
245 250
<210> 27
<211> 2486
<212> DNA
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和IL35结构域的融合蛋白的核苷酸表达盒
<400> 27
aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60
gataccaccg gtatgcacgt ggcccagcct gctgtggtac tggccagcag ccgaggcatc 120
gccagctttg tgtgtgagta tgcatctcca ggcaaagcca ctgaggtccg ggtgacagtg 180
cttcggcagg ctgacagcca ggtgactgaa gtctgtgcgg caacctacat gatggggaat 240
gagttgacct tcctagatga ttccatctgc acgggcacct ccagtggaaa tcaagtgaac 300
ctcactatcc aaggactgag ggccatggac acgggactct acatctgcaa ggtggagctc 360
atgtacccac cgccatacta cctgggcata ggcaacggaa cccagattta tgtaattgat 420
ccagaaccgt gcccagattc tgacctcgag cccaaatctt ctgacaaaac tcacacatgc 480
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 540
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 600
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 660
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 720
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 780
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 840
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 900
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 960
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1020
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1080
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1140
cagaggcaca acaattcttc cctgaataca agaactcaga aagcacgtca ttctccgaat 1200
tctaggaaag ggcccccagc agctctgaca ctgccccggg tgcaatgccg agcctctcgg 1260
tacccgatcg ccgtggattg ctcctggacc ctgccgcctg ctccaaactc caccagcccc 1320
gtgtccttca ttgccacgta caggctcggc atggctgccc ggggccacag ctggccctgc 1380
ctgcagcaga cgccaacgtc caccagctgc accatcacgg atgtccagct gttctccatg 1440
gctccctacg tgctcaatgt caccgccgtc cacccctggg gctccagcag cagcttcgtg 1500
cctttcataa cagagcacat catcaagccc gaccctccag aaggcgtgcg cctaagcccc 1560
ctcgctgagc gccagctaca ggtgcagtgg gagcctcccg ggtcctggcc cttcccagag 1620
atcttctcac tgaagtactg gatccgttac aagcgtcagg gagctgcgcg cttccaccgg 1680
gtggggccca ttgaagccac gtccttcatc ctcagggctg tgcggccccg agccaggtac 1740
tacgtccaag tggcggctca ggacctcaca gactacgggg aactgagtga ctggagtctc 1800
cccgccactg ccacaatgag cctgggcaag ggcggaggct cagggggtgg ctctggaggc 1860
ggctccggag ggggatctag aaacctcccc gtggccactc cagacccagg aatgttccca 1920
tgccttcacc actcccaaaa cctgctgagg gccgtcagca acatgctcca gaaggccaga 1980
caaactctag aattttaccc ttgcacttct gaagagattg atcatgaaga tatcacaaaa 2040
gataaaacca gcacagtgga ggcctgttta ccattggaat taaccaagaa tgagagttgc 2100
ctaaattcca gagagacctc tttcataact aatgggagtt gcctggcctc cagaaagacc 2160
tcttttatga tggccctgtg ccttagtagt atttatgaag acttgaagat gtaccaggtg 2220
gagttcaaga ccatgaatgc aaagcttctg atggatccta agaggcagat ctttctagat 2280
caaaacatgc tggcagttat tgatgagctg atgcaggccc tgaatttcaa cagtgagact 2340
gtgccacaaa aatcctccct tgaagaaccg gatttttata aaactaaaat caagctctgc 2400
atacttcttc atgctttcag aattcgggca gtgactattg atagagtgat gagctatctg 2460
aatgcttcct aatctagagc ggccgc 2486
<210> 28
<211> 799
<212> PRT
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和IL35结构域的融合蛋白
<400> 28
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr
355 360 365
Gln Lys Ala Arg His Ser Pro Asn Ser Arg Lys Gly Pro Pro Ala Ala
370 375 380
Leu Thr Leu Pro Arg Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala
385 390 395 400
Val Asp Cys Ser Trp Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro
405 410 415
Val Ser Phe Ile Ala Thr Tyr Arg Leu Gly Met Ala Ala Arg Gly His
420 425 430
Ser Trp Pro Cys Leu Gln Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile
435 440 445
Thr Asp Val Gln Leu Phe Ser Met Ala Pro Tyr Val Leu Asn Val Thr
450 455 460
Ala Val His Pro Trp Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr
465 470 475 480
Glu His Ile Ile Lys Pro Asp Pro Pro Glu Gly Val Arg Leu Ser Pro
485 490 495
Leu Ala Glu Arg Gln Leu Gln Val Gln Trp Glu Pro Pro Gly Ser Trp
500 505 510
Pro Phe Pro Glu Ile Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg
515 520 525
Gln Gly Ala Ala Arg Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser
530 535 540
Phe Ile Leu Arg Ala Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val
545 550 555 560
Ala Ala Gln Asp Leu Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser Leu
565 570 575
Pro Ala Thr Ala Thr Met Ser Leu Gly Lys Gly Gly Gly Ser Gly Gly
580 585 590
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Arg Asn Leu Pro Val Ala
595 600 605
Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu
610 615 620
Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu
625 630 635 640
Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys
645 650 655
Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys
660 665 670
Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly
675 680 685
Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu
690 695 700
Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr
705 710 715 720
Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp
725 730 735
Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe
740 745 750
Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe
755 760 765
Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile
770 775 780
Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
785 790 795
<210> 29
<211> 200
<212> PRT
<213> 智人(Homo sapiens)
<400> 29
Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro
1 5 10 15
Lys Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala
20 25 30
Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro
35 40 45
Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys
50 55 60
Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro
65 70 75 80
Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys
85 90 95
Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser
100 105 110
Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys Ile
115 120 125
Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr Ala Thr Ser
130 135 140
Ser Pro Gly Gln Arg Val Gln Lys Gly Gly Thr Glu Ser Gln Asp Thr
145 150 155 160
Leu Cys Gln Asn Cys Pro Pro Gly Thr Phe Ser Pro Asn Gly Thr Leu
165 170 175
Glu Glu Cys Gln His Gln Thr Lys Cys Ser Trp Leu Val Thr Lys Ala
180 185 190
Gly Ala Gly Thr Ser Ser Ser His
195 200
<210> 30
<211> 1707
<212> DNA
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和HVEM胞外域的融合蛋白的核苷酸表达盒
<400> 30
aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60
gataccaccg gtatgcacgt ggcccagcct gctgtggtac tggccagcag ccgaggcatc 120
gccagctttg tgtgtgagta tgcatctcca ggcaaagcca ctgaggtccg ggtgacagtg 180
cttcggcagg ctgacagcca ggtgactgaa gtctgtgcgg caacctacat gatggggaat 240
gagttgacct tcctagatga ttccatctgc acgggcacct ccagtggaaa tcaagtgaac 300
ctcactatcc aaggactgag ggccatggac acgggactct acatctgcaa ggtggagctc 360
atgtacccac cgccatacta cctgggcata ggcaacggaa cccagattta tgtaattgat 420
ccagaaccgt gcccagattc tgacctcgag cccaaatctt ctgacaaaac tcacacatgc 480
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 540
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 600
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 660
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 720
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 780
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 840
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 900
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 960
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1020
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1080
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1140
cagaggcaca acaattcttc cctgaataca agaactcaga aagcacgtca ttctccgaat 1200
tctctgccgt cctgcaagga ggacgagtac ccagtgggct ccgagtgctg ccccaagtgc 1260
agtccaggtt atcgtgtgaa ggaggcctgc ggggagctga cgggcacagt gtgtgaaccc 1320
tgccctccag gcacctacat tgcccacctc aatggcctaa gcaagtgtct gcagtgccaa 1380
atgtgtgacc cagccatggg cctgcgcgcg agccggaact gctccaggac agagaacgcc 1440
gtgtgtggct gcagcccagg ccacttctgc atcgtccagg acggggacca ctgcgccgcg 1500
tgccgcgctt acgccacctc cagcccgggc cagagggtgc agaagggagg caccgagagt 1560
caggacaccc tgtgtcagaa ctgccccccg gggaccttct ctcccaatgg gaccctggag 1620
gaatgtcagc accagaccaa gtgcagctgg ctggtgacga aggccggagc tgggaccagc 1680
agctcccact aaatctagag cggccgc 1707
<210> 31
<211> 539
<212> PRT
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和HVEM胞外域的融合蛋白
<400> 31
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr
355 360 365
Gln Lys Ala Arg His Ser Pro Asn Ser Leu Pro Ser Cys Lys Glu Asp
370 375 380
Glu Tyr Pro Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr
385 390 395 400
Arg Val Lys Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro
405 410 415
Cys Pro Pro Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys
420 425 430
Leu Gln Cys Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg
435 440 445
Asn Cys Ser Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His
450 455 460
Phe Cys Ile Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr
465 470 475 480
Ala Thr Ser Ser Pro Gly Gln Arg Val Gln Lys Gly Gly Thr Glu Ser
485 490 495
Gln Asp Thr Leu Cys Gln Asn Cys Pro Pro Gly Thr Phe Ser Pro Asn
500 505 510
Gly Thr Leu Glu Glu Cys Gln His Gln Thr Lys Cys Ser Trp Leu Val
515 520 525
Thr Lys Ala Gly Ala Gly Thr Ser Ser Ser His
530 535
<210> 32
<211> 238
<212> PRT
<213> 智人(Homo sapiens)
<400> 32
Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu
1 5 10 15
Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr
20 25 30
Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu
35 40 45
Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile
50 55 60
Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser
65 70 75 80
Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn
85 90 95
Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
100 105 110
Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val
115 120 125
Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val
130 135 140
Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr
145 150 155 160
Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser
165 170 175
Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn
180 185 190
Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr
195 200 205
Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu
210 215 220
Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg
225 230 235
<210> 33
<211> 230
<212> PRT
<213> 智人(Homo sapiens)
<400> 33
Met Ile Phe Leu Leu Leu Met Leu Ser Leu Glu Leu Gln Leu His Gln
1 5 10 15
Ile Ala Ala Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile
20 25 30
Glu His Gly Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser
35 40 45
His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn
50 55 60
Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu
65 70 75 80
Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp
85 90 95
Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr
100 105 110
Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr
115 120 125
His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln
130 135 140
Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val
145 150 155 160
Pro Ala Asn Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val
165 170 175
Thr Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys
180 185 190
Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp
195 200 205
Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu Leu His
210 215 220
Ile Phe Ile Pro Phe Cys
225 230
<210> 34
<211> 1823
<212> DNA
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和PDL2结构域的融合蛋白的核苷酸表达盒
<400> 34
aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60
gataccaccg gtatgcacgt ggcccagcct gctgtggtac tggccagcag ccgaggcatc 120
gccagctttg tgtgtgagta tgcatctcca ggcaaagcca ctgaggtccg ggtgacagtg 180
cttcggcagg ctgacagcca ggtgactgaa gtctgtgcgg caacctacat gatggggaat 240
gagttgacct tcctagatga ttccatctgc acgggcacct ccagtggaaa tcaagtgaac 300
ctcactatcc aaggactgag ggccatggac acgggactct acatctgcaa ggtggagctc 360
atgtacccac cgccatacta cctgggcata ggcaacggaa cccagattta tgtaattgat 420
ccagaaccgt gcccagattc tgacctcgag cccaaatctt ctgacaaaac tcacacatgc 480
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 540
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 600
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 660
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 720
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 780
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 840
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 900
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 960
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1020
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1080
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1140
cagaggcaca acaattcttc cctgaataca agaactcaga aagcacgtca ttctccgaat 1200
tctttattca cagtgacagt ccctaaggaa ctgtacataa tagagcatgg cagcaatgtg 1260
accctggaat gcaactttga cactggaagt catgtgaacc ttggagcaat aacagccagt 1320
ttgcaaaagg tggaaaatga tacatcccca caccgtgaaa gagccacttt gctggaggag 1380
cagctgcccc tagggaaggc ctcgttccac atacctcaag tccaagtgag ggacgaagga 1440
cagtaccaat gcataatcat ctatggggtc gcctgggact acaagtacct gaccctgaaa 1500
gtcaaagctt cctacaggaa aataaacact cacatcctaa aggttccaga aacagatgag 1560
gtagagctca cctgccaggc tacaggttat cctctggcag aagtatcctg gccaaacgtc 1620
agcgttcctg ccaacaccag ccactccagg acccctgaag gcctctacca ggtcaccagt 1680
gttctgcgcc taaagccacc ccctggcaga aacttcagct gtgtgttctg gaatactcac 1740
gtgagggaac ttactttggc cagcattgac cttcaaagtc agatggaacc caggacccat 1800
ccaacttaat ctagagcggc cgc 1823
<210> 35
<211> 578
<212> PRT
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和PDL2结构域的融合蛋白
<400> 35
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr
355 360 365
Gln Lys Ala Arg His Ser Pro Asn Ser Leu Phe Thr Val Thr Val Pro
370 375 380
Lys Glu Leu Tyr Ile Ile Glu His Gly Ser Asn Val Thr Leu Glu Cys
385 390 395 400
Asn Phe Asp Thr Gly Ser His Val Asn Leu Gly Ala Ile Thr Ala Ser
405 410 415
Leu Gln Lys Val Glu Asn Asp Thr Ser Pro His Arg Glu Arg Ala Thr
420 425 430
Leu Leu Glu Glu Gln Leu Pro Leu Gly Lys Ala Ser Phe His Ile Pro
435 440 445
Gln Val Gln Val Arg Asp Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr
450 455 460
Gly Val Ala Trp Asp Tyr Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser
465 470 475 480
Tyr Arg Lys Ile Asn Thr His Ile Leu Lys Val Pro Glu Thr Asp Glu
485 490 495
Val Glu Leu Thr Cys Gln Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser
500 505 510
Trp Pro Asn Val Ser Val Pro Ala Asn Thr Ser His Ser Arg Thr Pro
515 520 525
Glu Gly Leu Tyr Gln Val Thr Ser Val Leu Arg Leu Lys Pro Pro Pro
530 535 540
Gly Arg Asn Phe Ser Cys Val Phe Trp Asn Thr His Val Arg Glu Leu
545 550 555 560
Thr Leu Ala Ser Ile Asp Leu Gln Ser Gln Met Glu Pro Arg Thr His
565 570 575
Pro Thr
<210> 36
<400> 36
000
<210> 37
<220>
<223> 具有CTLA4胞外域和HVEM胞外域的融合蛋白的核苷酸表达盒
<400> 37
000
<210> 38
<220>
<223> 具有CTLA4胞外域和HVEM胞外域的融合蛋白
<400> 38
000
<210> 39
<211> 164
<212> PRT
<213> 智人(Homo sapiens)
<400> 39
Met Ala Gln His Gly Ala Met Gly Ala Phe Arg Ala Leu Cys Gly Leu
1 5 10 15
Ala Leu Leu Cys Ala Leu Ser Leu Gly Gln Arg Pro Thr Gly Gly Pro
20 25 30
Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly Thr Asp Ala Arg
35 40 45
Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp Tyr Pro Gly Glu
50 55 60
Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln Pro Glu Phe His
65 70 75 80
Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His Pro Cys Pro Pro
85 90 95
Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe Gly Phe Gln Cys
100 105 110
Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His Glu Gly His Cys
115 120 125
Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu Thr Val Phe Pro
130 135 140
Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly Ser Pro Pro Ala
145 150 155 160
Glu Pro Leu Gly
<210> 40
<211> 255
<212> PRT
<213> 智人(Homo sapiens)
<400> 40
Met Ala Gln His Gly Ala Met Gly Ala Phe Arg Ala Leu Cys Gly Leu
1 5 10 15
Ala Leu Leu Cys Ala Leu Ser Leu Gly Gln Arg Pro Thr Gly Gly Pro
20 25 30
Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly Thr Asp Ala Arg
35 40 45
Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp Tyr Pro Gly Glu
50 55 60
Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln Pro Glu Phe His
65 70 75 80
Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His Pro Cys Pro Pro
85 90 95
Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe Gly Phe Gln Cys
100 105 110
Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His Glu Gly His Cys
115 120 125
Lys Pro Trp Thr Asp Cys Cys Trp Arg Cys Arg Arg Arg Pro Lys Thr
130 135 140
Pro Glu Ala Ala Ser Ser Pro Arg Lys Ser Gly Ala Ser Asp Arg Gln
145 150 155 160
Arg Arg Arg Gly Gly Trp Glu Thr Cys Gly Cys Glu Pro Gly Arg Pro
165 170 175
Pro Gly Pro Pro Thr Ala Ala Ser Pro Ser Pro Gly Ala Pro Gln Ala
180 185 190
Ala Gly Ala Leu Arg Ser Ala Leu Gly Arg Ala Leu Leu Pro Trp Gln
195 200 205
Gln Lys Trp Val Gln Glu Gly Gly Ser Asp Gln Arg Pro Gly Pro Cys
210 215 220
Ser Ser Ala Ala Ala Ala Gly Pro Cys Arg Arg Glu Arg Glu Thr Gln
225 230 235 240
Ser Trp Pro Pro Ser Ser Leu Ala Gly Pro Asp Gly Val Gly Ser
245 250 255
<210> 41
<211> 1631
<212> DNA
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和GITR结构域的融合蛋白的核苷酸表达盒
<400> 41
aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60
gataccaccg gtatgcacgt ggcccagcct gctgtggtac tggccagcag ccgaggcatc 120
gccagctttg tgtgtgagta tgcatctcca ggcaaagcca ctgaggtccg ggtgacagtg 180
cttcggcagg ctgacagcca ggtgactgaa gtctgtgcgg caacctacat gatggggaat 240
gagttgacct tcctagatga ttccatctgc acgggcacct ccagtggaaa tcaagtgaac 300
ctcactatcc aaggactgag ggccatggac acgggactct acatctgcaa ggtggagctc 360
atgtacccac cgccatacta cctgggcata ggcaacggaa cccagattta tgtaattgat 420
ccagaaccgt gcccagattc tgacctcgag cccaaatctt ctgacaaaac tcacacatgc 480
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 540
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 600
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 660
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 720
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 780
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 840
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 900
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 960
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1020
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1080
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1140
cagaggcaca acaattcttc cctgaataca agaactcaga aagcacgtca ttctccgaat 1200
tctcagcgcc ccaccggggg tcccgggtgc ggccctgggc gcctcctgct tgggacggga 1260
acggacgcgc gctgctgccg ggttcacacg acgcgctgct gccgcgatta cccgggcgag 1320
gagtgctgtt ccgagtggga ctgcatgtgt gtccagcctg aattccactg cggagaccct 1380
tgctgcacga cctgccggca ccacccttgt cccccaggcc agggggtaca gtcccagggg 1440
aaattcagtt ttggcttcca gtgtatcgac tgtgcctcgg ggaccttctc cgggggccac 1500
gaaggccact gcaaaccttg gacagactgc acccagttcg ggtttctcac tgtgttccct 1560
gggaacaaga cccacaacgc tgtgtgcgtc ccagggtccc ccccggcaga gccgcttggg 1620
taagcggccg c 1631
<210> 42
<211> 516
<212> PRT
<213> 人工序列
<220>
<223> 具有CTLA4胞外域和GITR结构域的融合蛋白
<400> 42
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr
355 360 365
Gln Lys Ala Arg His Ser Pro Asn Ser Gln Arg Pro Thr Gly Gly Pro
370 375 380
Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly Thr Asp Ala Arg
385 390 395 400
Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp Tyr Pro Gly Glu
405 410 415
Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln Pro Glu Phe His
420 425 430
Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His Pro Cys Pro Pro
435 440 445
Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe Gly Phe Gln Cys
450 455 460
Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His Glu Gly His Cys
465 470 475 480
Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu Thr Val Phe Pro
485 490 495
Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly Ser Pro Pro Ala
500 505 510
Glu Pro Leu Gly
515
<210> 43
<211> 2
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 43
Asn Ser
1
<210> 44
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 44
Ser Cys Pro Pro Cys Pro
1 5
<210> 45
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 45
Gly Gly Gly Gly Ser Gly Asn Ser
1 5
<210> 46
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 46
Gly Cys Pro Pro Cys Pro Asn Ser
1 5
<210> 47
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 47
Gly Ser Pro Pro Ser Pro Asn Ser
1 5
<210> 48
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 48
Gly Ser Pro Pro Ser Pro Asn Ser
1 5
<210> 49
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 49
Gly Cys Pro Pro Cys Pro Asn Ser
1 5
<210> 50
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 50
Gly Cys Pro Pro Cys Pro Asn Ser
1 5
<210> 51
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 51
Gly Cys Pro Pro Cys Pro Gly Asn Ser
1 5
<210> 52
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 52
Gly Cys Pro Pro Cys Pro Ala Asn Ser
1 5
<210> 53
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 53
Gly Cys Pro Pro Cys Pro Ala Asn Ser
1 5
<210> 54
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 54
Glu Glu Glu Glu Asp Glu Gly Asn Ser
1 5
<210> 55
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 55
Asn Tyr Gly Gly Gly Gly Ser Gly Asn Ser
1 5 10
<210> 56
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 56
Val Ser Glu Arg Pro Phe Pro Pro Asn Ser
1 5 10
<210> 57
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 57
Glu Pro Lys Ser Cys Asp Lys Thr Cys Cys Pro
1 5 10
<210> 58
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 58
Ser Gln Pro Glu Ile Val Pro Ile Ser Asn Ser
1 5 10
<210> 59
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 59
Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Asn Ser
1 5 10
<210> 60
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 60
Lys Ala Asp Phe Leu Thr Pro Ser Ile Gly Asn Ser
1 5 10
<210> 61
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 61
Gln Met Asn Ser Glu Leu Ser Val Leu Ala Asn Ser
1 5 10
<210> 62
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 62
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Cys Pro
1 5 10
<210> 63
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 63
Glu Pro Lys Ser Cys Asp Lys Thr Cys Pro Pro Cys Pro
1 5 10
<210> 64
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 64
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asn Ser
1 5 10
<210> 65
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 65
Gly Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Asn Ser
1 5 10
<210> 66
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 66
Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Gly Asn Ser
1 5 10
<210> 67
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 67
Gly Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Asn Ser
1 5 10
<210> 68
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 68
Gly Gly Gly Ala Ser Cys Pro Pro Cys Pro Gly Asn Ser
1 5 10
<210> 69
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 69
Gly Gly Gly Ala Ser Cys Pro Pro Cys Ala Gly Asn Ser
1 5 10
<210> 70
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 70
Gly Gly Gly Ala Ser Cys Pro Pro Cys Ala Gly Asn Ser
1 5 10
<210> 71
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 71
Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asn Ser
1 5 10 15
<210> 72
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 72
Leu Ser Val Lys Ala Asp Phe Leu Thr Pro Ser Ile Gly Asn Ser
1 5 10 15
<210> 73
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 73
Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Gly Asn Ser
1 5 10 15
<210> 74
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 74
Leu Lys Ile Gln Glu Arg Val Ser Lys Pro Lys Ile Ser Asn Ser
1 5 10 15
<210> 75
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 75
Leu Asp Val Ser Glu Arg Pro Phe Pro Pro His Ile Gln Asn Ser
1 5 10 15
<210> 76
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 76
Arg Glu Gln Leu Ala Glu Val Thr Leu Ser Leu Lys Ala Asn Ser
1 5 10 15
<210> 77
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 77
Arg Ile His Gln Met Asn Ser Glu Leu Ser Val Leu Ala Asn Ser
1 5 10 15
<210> 78
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 78
Asp Thr Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Ser Asn Ser
1 5 10 15
<210> 79
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 79
Leu Pro Pro Glu Thr Gln Glu Ser Gln Glu Val Thr Leu Asn Ser
1 5 10 15
<210> 80
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 80
Arg Ile His Leu Asn Val Ser Glu Arg Pro Phe Pro Pro Asn Ser
1 5 10 15
<210> 81
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 81
Leu Ser Val Lys Ala Asp Phe Leu Thr Pro Ser Ile Gly Asn Ser
1 5 10 15
<210> 82
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 82
Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Gly Asn Ser
1 5 10 15
<210> 83
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 83
Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Gly Asn Ser
1 5 10 15
<210> 84
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 84
Arg Ile His Gln Met Asn Ser Glu Leu Ser Val Leu Ala Asn Ser
1 5 10 15
<210> 85
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 85
Lys Pro Phe Phe Thr Cys Gly Ser Ala Asp Thr Cys Pro Asn Ser
1 5 10 15
<210> 86
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 86
Lys Pro Phe Phe Thr Cys Gly Ser Ala Asp Thr Cys Pro Asn Ser
1 5 10 15
<210> 87
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 87
Gln Tyr Asn Cys Pro Gly Gln Tyr Thr Phe Ser Met Pro Asn Ser
1 5 10 15
<210> 88
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 88
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 89
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 89
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 90
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 90
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Ser
1 5 10 15
<210> 91
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 91
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Cys Ser
1 5 10 15
<210> 92
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 92
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 93
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 93
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser Ser
1 5 10 15
<210> 94
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 94
Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro
1 5 10 15
<210> 95
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 95
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Ser
1 5 10 15
<210> 96
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 96
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Cys Ser
1 5 10 15
<210> 97
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 97
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Ser Ser
1 5 10 15
<210> 98
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 98
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Cys Pro
1 5 10 15
<210> 99
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 99
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro
1 5 10 15
<210> 100
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 100
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Cys Pro
1 5 10 15
<210> 101
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 101
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser Pro
1 5 10 15
<210> 102
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 102
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Ser Pro
1 5 10 15
<210> 103
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 103
Gly Gly Gly Gly Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 104
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 104
Glu Pro Lys Ser Cys Gly Gly Gly Gly Gly Cys Pro Pro Cys Pro
1 5 10 15
<210> 105
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 105
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Gly Gly Cys Pro
1 5 10 15
<210> 106
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 106
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Gly
1 5 10 15
<210> 107
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 107
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Ser Pro
1 5 10 15
<210> 108
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 108
Glu Pro Lys Ser Cys Asp Lys Cys His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 109
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 109
Glu Pro Lys Ser Cys Asp Lys Thr Cys Cys Cys Pro Pro Cys Pro
1 5 10 15
<210> 110
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 110
Glu Pro Lys Ser Cys Pro Pro Pro Pro Pro Cys Pro Pro Cys Pro
1 5 10 15
<210> 111
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 111
Pro Pro Pro Pro Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 112
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 112
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Trp Trp Cys Pro
1 5 10 15
<210> 113
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 113
Glu Pro Lys Ser Cys Asp Trp Trp His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 114
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 114
Glu Pro Lys Cys Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 115
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 115
Glu Pro Lys Ser Asp Cys Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 116
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 116
Glu Pro Lys Ser Asp Cys Trp Trp His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 117
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 117
Glu Pro Lys Ser Cys Asp Phe Phe His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 118
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 118
Glu Pro Lys Ser Cys Asp Trp Trp Trp Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 119
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 119
Glu Pro Lys Ser Cys Trp Trp Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 120
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 120
Glu Pro Trp Trp Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 121
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 121
Ser Gln Pro Glu Ile Val Pro Ile Ser Cys Pro Pro Cys Pro Asn Ser
1 5 10 15
<210> 122
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 122
Thr Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys
1 5 10 15
Pro
<210> 123
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 123
Glu Pro Lys Ser Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Asn
1 5 10 15
Ser
<210> 124
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 124
Glu Pro Lys Ser Thr Asp Lys Thr His Thr Ser Pro Pro Ser Pro Asn
1 5 10 15
Ser
<210> 125
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 125
Glu Pro Lys Ser Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Asn
1 5 10 15
Ser
<210> 126
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 126
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Asn
1 5 10 15
Ser
<210> 127
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 127
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Gly Gly Gly Pro
1 5 10 15
Cys Pro
<210> 128
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 128
Glu Pro Lys Ser Cys Asp Gly Gly Gly Lys Thr His Thr Cys Pro Pro
1 5 10 15
Cys Pro
<210> 129
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 129
Glu Pro Lys Ser Cys Asp Pro Pro Pro Lys Thr His Thr Cys Pro Pro
1 5 10 15
Cys Pro
<210> 130
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 130
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Pro Pro Pro
1 5 10 15
Cys Pro
<210> 131
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 131
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Asn Ser
<210> 132
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 132
Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser Asn Ser
<210> 133
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 133
Leu Ser Val Lys Ala Asp Phe Leu Thr Pro Ser Ile Ser Pro Pro Cys
1 5 10 15
Pro Asn Ser
<210> 134
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 134
Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Ser Cys Pro Pro Cys
1 5 10 15
Pro Asn Ser
<210> 135
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 135
Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gln Lys Ala
1 5 10 15
Arg His Ser Pro
20
<210> 136
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 136
Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Ser Cys Pro Pro
1 5 10 15
Cys Pro Asn Ser
20
<210> 137
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 137
Leu Lys Ile Gln Glu Arg Val Ser Lys Pro Lys Ile Ser Cys Pro Pro
1 5 10 15
Cys Pro Asn Ser
20
<210> 138
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 138
Arg Glu Gln Leu Ala Glu Val Thr Leu Ser Leu Lys Ala Cys Pro Pro
1 5 10 15
Cys Pro Asn Ser
20
<210> 139
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 139
Arg Ile His Gln Met Asn Ser Glu Leu Ser Val Leu Ala Cys Pro Pro
1 5 10 15
Cys Pro Asn Ser
20
<210> 140
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 140
Arg Ile His Leu Asn Val Ser Glu Arg Pro Phe Pro Pro Cys Pro Pro
1 5 10 15
Cys Pro Asn Ser
20
<210> 141
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 141
Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr
1 5 10 15
Cys Pro Asn Ser
20
<210> 142
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 142
Glu Glu Glu Glu Asp Glu Glu Asp Glu Glu Asp Glu Glu Glu Glu Glu
1 5 10 15
Asp Gly Asn Ser
20
<210> 143
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 143
Leu Asp Val Ser Glu Arg Pro Phe Pro Pro His Ile Gln Ser Cys Pro
1 5 10 15
Pro Cys Pro Asn Ser
20
<210> 144
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 144
Asp Thr Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Ser Cys Pro
1 5 10 15
Pro Cys Pro Asn Ser
20
<210> 145
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 145
Leu Pro Pro Glu Thr Gln Glu Ser Gln Glu Val Thr Leu Ser Cys Pro
1 5 10 15
Pro Cys Pro Asn Ser
20
<210> 146
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 146
Glu Pro Ala Phe Thr Pro Gly Pro Asn Ile Glu Leu Gln Lys Asp Ser
1 5 10 15
Asp Cys Pro Asn Ser
20
<210> 147
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 147
Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gln Lys Ala Arg
1 5 10 15
His Cys Pro Asn Ser
20
<210> 148
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 148
Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gln Lys Ala Arg
1 5 10 15
His Ser Pro Asn Ser
20
<210> 149
<211> 36
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 149
Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
20 25 30
Gly Ser Asn Ser
35
<210> 150
<211> 122
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 150
Arg Thr Arg Tyr Leu Gln Val Ser Gln Gln Leu Gln Gln Thr Asn Arg
1 5 10 15
Val Leu Glu Val Thr Asn Ser Ser Leu Arg Gln Gln Leu Arg Leu Lys
20 25 30
Ile Thr Gln Leu Gly Gln Ser Ala Glu Asp Leu Gln Gly Ser Arg Arg
35 40 45
Glu Leu Ala Gln Ser Gln Glu Ala Leu Gln Val Glu Gln Arg Ala His
50 55 60
Gln Ala Ala Glu Gly Gln Leu Gln Ala Cys Gln Ala Asp Arg Gln Lys
65 70 75 80
Thr Lys Glu Thr Leu Gln Ser Glu Glu Gln Gln Arg Arg Ala Leu Glu
85 90 95
Gln Lys Leu Ser Asn Met Glu Asn Arg Leu Lys Pro Phe Phe Thr Cys
100 105 110
Gly Ser Ala Asp Thr Cys Cys Pro Asn Ser
115 120
<210> 151
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 151
Gly Gly Gly Ala Ser Cys Pro Pro Cys Ala Gly Asn Ser
1 5 10
<210> 152
<211> 39
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 152
Asn Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
1 5 10 15
Gly Ser Gly Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
20 25 30
Gly Gly Gly Ser Gly Asn Ser
35
<210> 153
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 153
Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser Gly Asn Ser
20
<210> 154
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 154
Ser Pro Pro Ser Pro Asn Ser
1 5
<210> 155
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 155
Glu Pro Thr Ser Thr Asp Lys Thr His Thr Ser Pro Pro Ser Pro Asn
1 5 10 15
Ser
<210> 156
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 156
Glu Pro Thr Ser Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Asn
1 5 10 15
Ser
<210> 157
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 157
Leu Ser Val Lys Ala Asp Phe Leu Thr Pro Ser Ile Ser Pro Pro Cys
1 5 10 15
Pro Asn Ser
<210> 158
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 158
Gly Gly Gly Ala Ser Cys Pro Pro Cys Ala Asn Ser
1 5 10
<210> 159
<211> 22
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 159
Arg Thr Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gln Lys
1 5 10 15
Ala Arg His Ser Gly His
20
<210> 160
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 160
Arg Thr Arg Tyr Leu Gln Val Ser Gln Gln Leu Gln Gln Thr Asn Arg
1 5 10 15
Val Leu Glu Val Thr Asn Ser Ser Leu Arg Gln Gln Leu Arg Leu Lys
20 25 30
Ile Thr Gln Leu Gly Gln Ser Ala Glu Asp Leu Gln Gly Ser Arg Arg
35 40 45
Glu Leu Ala Gln Ser Gln Glu Ala Leu Gln Val Glu Gln Arg Ala His
50 55 60
Gln Ala Ala Glu Gly Gln Leu Gln Ala Cys Gln Ala Asp Arg Gln Lys
65 70 75 80
Thr Lys Glu Thr Leu Gln Ser Glu Glu Gln Gln Arg Arg Ala Leu Glu
85 90 95
Gln Lys Leu Ser Asn Met Glu Asn Arg Leu Lys Pro Phe Phe Thr Cys
100 105 110
Gly Ser Ala Asp Thr Cys
115
<210> 161
<211> 35
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 161
Arg Thr Gln Ser Glu Glu Gln Gln Arg Arg Ala Leu Glu Gln Lys Leu
1 5 10 15
Ser Asn Met Glu Asn Arg Leu Lys Pro Phe Phe Thr Cys Gly Ser Ala
20 25 30
Asp Thr Cys
35
<210> 162
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 162
Arg Thr Glu Gln Lys Leu Ser Asn Met Glu Asn Arg Leu Lys Pro Phe
1 5 10 15
Phe Thr Cys Gly Ser Ala Asp Thr Cys
20 25
<210> 163
<211> 26
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 163
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 164
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 164
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Ser
1 5 10 15
<210> 165
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 165
Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 166
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 166
Gly Asp Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro
1 5 10 15
Cys Pro
<210> 167
<211> 22
<212> PRT
<213> 人工序列
<220>
<223> 异源前导序列
<400> 167
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly
20
<210> 168
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 异源前导序列
<400> 168
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
Asp Thr Thr Gly
20
<210> 169
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 编码异源前导序列的多核苷酸
<400> 169
atggaagcac cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60
<210> 170
<211> 1611
<212> DNA
<213> 人工序列
<220>
<223> 编码含H68接头的CTLA4::IL10融合蛋白的核苷酸序列
<400> 170
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa ctcattattc 1080
aaccaagaag ttcaaattcc cttgaccgaa agttacagcc cgaattctag cccaggccag 1140
ggcacccagt ctgagaacag ctgcacccac ttcccaggca acctgcctaa catgcttcga 1200
gatctccgag atgccttcag cagagtgaag actttctttc aaatgaagga tcagctggac 1260
aacttgttgt taaaggagtc cttgctggag gactttaagg gttacctggg ttgccaagcc 1320
ttgtctgaga tgatccagtt ttacctggag gaggtgatgc cccaagctga gaaccaagac 1380
ccagacatca aggcgcatgt gaactccctg ggggagaacc tgaagaccct caggctgagg 1440
ctacggcgct gtcatcgatt tcttccctgt gaaaacaaga gcaaggccgt ggagcaggtg 1500
aagaatgcct ttaataagct ccaagagaaa ggcatctaca aagccatgag tgagtttgac 1560
atcttcatca actacataga agcctacatg acaatgaaga tacgaaacta a 1611
<210> 171
<211> 536
<212> PRT
<213> 人工序列
<220>
<223> 含H68接头的CTLA4::IL10融合蛋白
<400> 171
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu
355 360 365
Thr Glu Ser Tyr Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln Ser
370 375 380
Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg
385 390 395 400
Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys
405 410 415
Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe
420 425 430
Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr
435 440 445
Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys
450 455 460
Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg
465 470 475 480
Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala
485 490 495
Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile
500 505 510
Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala
515 520 525
Tyr Met Thr Met Lys Ile Arg Asn
530 535
<210> 172
<211> 1614
<212> DNA
<213> 人工序列
<220>
<223> 编码含H75接头的CTLA4::IL10融合蛋白的核苷酸序列
<400> 172
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtca gaggcacaac 1080
aattcttccc tgaatacagg aactcagatg gcaggtcatt ctccgaattc tagcccaggc 1140
cagggcaccc agtctgagaa cagctgcacc cacttcccag gcaacctgcc taacatgctt 1200
cgagatctcc gagatgcctt cagcagagtg aagactttct ttcaaatgaa ggatcagctg 1260
gacaacttgt tgttaaagga gtccttgctg gaggacttta agggttacct gggttgccaa 1320
gccttgtctg agatgatcca gttttacctg gaggaggtga tgccccaagc tgagaaccaa 1380
gacccagaca tcaaggcgca tgtgaactcc ctgggggaga acctgaagac cctcaggctg 1440
aggctacggc gctgtcatcg atttcttccc tgtgaaaaca agagcaaggc cgtggagcag 1500
gtgaagaatg cctttaataa gctccaagag aaaggcatct acaaagccat gagtgagttt 1560
gacatcttca tcaactacat agaagcctac atgacaatga agatacgaaa ctaa 1614
<210> 173
<211> 537
<212> PRT
<213> 人工序列
<220>
<223> 含H75接头的CTLA4::IL10融合蛋白
<400> 173
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Gly Thr
355 360 365
Gln Met Ala Gly His Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln
370 375 380
Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu
385 390 395 400
Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met
405 410 415
Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp
420 425 430
Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe
435 440 445
Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile
450 455 460
Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu
465 470 475 480
Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys
485 490 495
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly
500 505 510
Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu
515 520 525
Ala Tyr Met Thr Met Lys Ile Arg Asn
530 535
<210> 174
<211> 1602
<212> DNA
<213> 人工序列
<220>
<223> 编码含H77接头的CTLA4::IL10融合蛋白的核苷酸序列
<400> 174
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa ctcattattc 1080
aaccaagaag ttcaaattcc cttgaccgaa ccgaattcta gcccaggcca gggcacccag 1140
tctgagaaca gctgcaccca cttcccaggc aacctgccta acatgcttcg agatctccga 1200
gatgccttca gcagagtgaa gactttcttt caaatgaagg atcagctgga caacttgttg 1260
ttaaaggagt ccttgctgga ggactttaag ggttacctgg gttgccaagc cttgtctgag 1320
atgatccagt tttacctgga ggaggtgatg ccccaagctg agaaccaaga cccagacatc 1380
aaggcgcatg tgaactccct gggggagaac ctgaagaccc tcaggctgag gctacggcgc 1440
tgtcatcgat ttcttccctg tgaaaacaag agcaaggccg tggagcaggt gaagaatgcc 1500
tttaataagc tccaagagaa aggcatctac aaagccatga gtgagtttga catcttcatc 1560
aactacatag aagcctacat gacaatgaag atacgaaact aa 1602
<210> 175
<211> 533
<212> PRT
<213> 人工序列
<220>
<223> 含H77接头的CTLA4::IL10融合蛋白
<400> 175
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu
355 360 365
Thr Glu Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser
370 375 380
Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg
385 390 395 400
Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu
405 410 415
Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr
420 425 430
Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu
435 440 445
Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val
450 455 460
Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg
465 470 475 480
Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln
485 490 495
Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala
500 505 510
Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr
515 520 525
Met Lys Ile Arg Asn
530
<210> 176
<211> 1593
<212> DNA
<213> 人工序列
<220>
<223> 编码含H78接头的CTLA4::IL10融合蛋白的核苷酸序列
<400> 176
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa ctcattattc 1080
aaccaagaag ttcaaattcc cccgaattct agcccaggcc agggcaccca gtctgagaac 1140
agctgcaccc acttcccagg caacctgcct aacatgcttc gagatctccg agatgccttc 1200
agcagagtga agactttctt tcaaatgaag gatcagctgg acaacttgtt gttaaaggag 1260
tccttgctgg aggactttaa gggttacctg ggttgccaag ccttgtctga gatgatccag 1320
ttttacctgg aggaggtgat gccccaagct gagaaccaag acccagacat caaggcgcat 1380
gtgaactccc tgggggagaa cctgaagacc ctcaggctga ggctacggcg ctgtcatcga 1440
tttcttccct gtgaaaacaa gagcaaggcc gtggagcagg tgaagaatgc ctttaataag 1500
ctccaagaga aaggcatcta caaagccatg agtgagtttg acatcttcat caactacata 1560
gaagcctaca tgacaatgaa gatacgaaac taa 1593
<210> 177
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 含H78接头的CTLA4::IL10融合蛋白
<400> 177
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Pro
355 360 365
Asn Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His
370 375 380
Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe
385 390 395 400
Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu
405 410 415
Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys
420 425 430
Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro
435 440 445
Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu
450 455 460
Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg
465 470 475 480
Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn
485 490 495
Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu
500 505 510
Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile
515 520 525
Arg Asn
530
<210> 178
<211> 1584
<212> DNA
<213> 人工序列
<220>
<223> 编码含H79接头的CTLA4::IL10融合蛋白的核苷酸序列
<400> 178
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa ctcattattc 1080
aaccaagaag ttccgaattc tagcccaggc cagggcaccc agtctgagaa cagctgcacc 1140
cacttcccag gcaacctgcc taacatgctt cgagatctcc gagatgcctt cagcagagtg 1200
aagactttct ttcaaatgaa ggatcagctg gacaacttgt tgttaaagga gtccttgctg 1260
gaggacttta agggttacct gggttgccaa gccttgtctg agatgatcca gttttacctg 1320
gaggaggtga tgccccaagc tgagaaccaa gacccagaca tcaaggcgca tgtgaactcc 1380
ctgggggaga acctgaagac cctcaggctg aggctacggc gctgtcatcg atttcttccc 1440
tgtgaaaaca agagcaaggc cgtggagcag gtgaagaatg cctttaataa gctccaagag 1500
aaaggcatct acaaagccat gagtgagttt gacatcttca tcaactacat agaagcctac 1560
atgacaatga agatacgaaa ctaa 1584
<210> 179
<211> 527
<212> PRT
<213> 人工序列
<220>
<223> 含H79接头的CTLA4::IL10融合蛋白
<400> 179
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Pro Asn Ser Ser
355 360 365
Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly
370 375 380
Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val
385 390 395 400
Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys
405 410 415
Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu
420 425 430
Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu
435 440 445
Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn
450 455 460
Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro
465 470 475 480
Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn
485 490 495
Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile
500 505 510
Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
515 520 525
<210> 180
<211> 1629
<212> DNA
<213> 人工序列
<220>
<223> 编码CTLA4::单IL10融合蛋白的核苷酸序列
<400> 180
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa ctcattattc 1080
aaccaagaag ttcaaattcc cttgaccgaa agttacagcc cgaattctag cccaggccag 1140
ggcacccagt ctgagaacag ctgcacccac ttcccaggca acctgcctaa catgcttcga 1200
gatctccgag atgccttcag cagagtgaag actttctttc aaatgaagga tcagctggac 1260
aacttgttgt taaaggagtc cttgctggag gactttaagg gttacctggg ttgccaagcc 1320
ttgtctgaga tgatccagtt ttacctggag gaggtgatgc cccaagctga gaaccaagac 1380
ccagacatca aggcgcatgt gaactccctg ggggagaacc tgaagaccct caggctgagg 1440
ctacggcgct gtcatcgatt tcttccctgt gaaaacggtg gtggatccgg cggtaagagc 1500
aaggccgtgg agcaggtgaa gaatgccttt aataagctcc aagagaaagg catctacaaa 1560
gccatgagtg agtttgacat cttcatcaac tacatagaag cctacatgac aatgaagata 1620
cgaaactaa 1629
<210> 181
<211> 542
<212> PRT
<213> 人工序列
<220>
<223> CTLA4::单IL10融合蛋白
<400> 181
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu
355 360 365
Thr Glu Ser Tyr Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln Ser
370 375 380
Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg
385 390 395 400
Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys
405 410 415
Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe
420 425 430
Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr
435 440 445
Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys
450 455 460
Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg
465 470 475 480
Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser
485 490 495
Gly Gly Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys
500 505 510
Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe
515 520 525
Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
530 535 540
<210> 182
<211> 2013
<212> DNA
<213> 人工序列
<220>
<223> 编码IL10::FUN1融合蛋白的核苷酸序列
<400> 182
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacat caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggtaact cattattcaa ccaagaagtt 1200
caaattccct tgaccgaaag ttacagcccg aattctgagg tccaactgca gcagtctgga 1260
cctgagctgg agaagcctgg cgcttcagtg aagatatcct gcaaggcttc tggttactca 1320
ttcactgact acaacatgaa ctgggtgaag cagagcaatg gaaagagcct tgagtggatt 1380
ggaaatattg atccttacta tggtggtact agttacaatc agaagttcaa gggcaaggcc 1440
acattgactg tagacaaatc ctccagcaca gcctacatgc agctcaacag cctgacatct 1500
gaagactctg cagtctattt ctgtgcaaga tgggactata ggtacgacga cgggagggct 1560
tactatgtta tggacttctg gggtcaagga acctcagtca ccgtctcctc agggggtgga 1620
ggctctggtg gcggtggctc tggcggaggt ggatccggtg gcggcggatc tgagctccag 1680
atgacccagt ctccatcatc tctggctgcg tctgcaggag aaaaggtcac tatgagctgt 1740
aagtccagtc aaagtgtttt atacagttca aatcagaaga actacttggc ctggtaccag 1800
cagaaaccag ggcagtctcc taaactgctg atctactggg catccactag ggaatctggt 1860
gtccctgatc gcttcacagg cagtggatct gggacacatt ttactctgac cgtcagcagt 1920
gtgcaagctg aagacctggc agtttattac tgtcatcaat acctctactc gtggacgttc 1980
ggtggaggca ccaacctgga aatcaaacgg taa 2013
<210> 183
<211> 670
<212> PRT
<213> 人工序列
<220>
<223> IL10::FUN1融合蛋白
<400> 183
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val
385 390 395 400
Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Glu Val Gln Leu
405 410 415
Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser Val Lys Ile
420 425 430
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Asn Met Asn Trp
435 440 445
Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile Gly Asn Ile Asp
450 455 460
Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala
465 470 475 480
Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Asn
485 490 495
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Trp Asp
500 505 510
Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met Asp Phe Trp Gly
515 520 525
Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
530 535 540
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln
545 550 555 560
Met Thr Gln Ser Pro Ser Ser Leu Ala Ala Ser Ala Gly Glu Lys Val
565 570 575
Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Gln
580 585 590
Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys
595 600 605
Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg
610 615 620
Phe Thr Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr Val Ser Ser
625 630 635 640
Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys His Gln Tyr Leu Tyr
645 650 655
Ser Trp Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys Arg
660 665 670
<210> 184
<211> 1983
<212> DNA
<213> 人工序列
<220>
<223> 编码IL10::3D1融合蛋白的核苷酸序列
<400> 184
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacat caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggtaact cattattcaa ccaagaagtt 1200
caaattccct tgaccgaaag ttacagcccg aattctcagg tccagctgca gcagtctggg 1260
cctgagctgg tgaggcctgg ggaatcagtg aagatttcct gcaagggttc cggctacaca 1320
ttcactgatt atgctataca gtgggtgaag cagagtcatg caaagagtct agagtggatt 1380
ggagttatta atatttacta tgataataca aactacaacc agaagtttaa gggcaaggcc 1440
acaatgactg tagacaaatc ctccagcaca gcctatatgg aacttgccag attgacatct 1500
gaggattctg ccatctatta ctgtgcaaga gcggcctggt atatggacta ctggggtcaa 1560
ggaacctcag tcaccgtctc ctcagggggt ggaggctctg gtggcggtgg ctctggcgga 1620
ggtggatccg gtggcggcgg atctgacatt gtgctgtcac agtctccatc ctccctggct 1680
gtgtcagcag gagagaaggt cactatgagc tgcaaatcca gtcagagtct gctcaacagt 1740
agaacccgag agaactactt ggcttggtac cagcagaaac cagggcagtc tcctaaactg 1800
ctgatctact gggcatccac tagggaatct ggggtccctg atcgcttcac aggcagtgga 1860
tctgggacag atttcactct caccatcagc agtgtgcagg ctgaagacct ggcagtttat 1920
tactgcacgc aatcttataa tctttacacg ttcggagggg ggaccaagct ggaaataaaa 1980
taa 1983
<210> 185
<211> 660
<212> PRT
<213> 人工序列
<220>
<223> IL10::3D1融合蛋白
<400> 185
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val
385 390 395 400
Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Gln Val Gln Leu
405 410 415
Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Glu Ser Val Lys Ile
420 425 430
Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala Ile Gln Trp
435 440 445
Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile Gly Val Ile Asn
450 455 460
Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala
465 470 475 480
Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ala
485 490 495
Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg Ala Ala
500 505 510
Trp Tyr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Asp Ile Val Leu Ser Gln Ser Pro Ser Ser Leu Ala
545 550 555 560
Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser
565 570 575
Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr Leu Ala Trp Tyr Gln Gln
580 585 590
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
595 600 605
Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
610 615 620
Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr
625 630 635 640
Tyr Cys Thr Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys
645 650 655
Leu Glu Ile Lys
660
<210> 186
<211> 2013
<212> DNA
<213> 人工序列
<220>
<223> 编码FUN1::IL10融合蛋白的核苷酸序列
<400> 186
gaggtccaac tgcagcagtc tggacctgag ctggagaagc ctggcgcttc agtgaagata 60
tcctgcaagg cttctggtta ctcattcact gactacaaca tgaactgggt gaagcagagc 120
aatggaaaga gccttgagtg gattggaaat attgatcctt actatggtgg tactagttac 180
aatcagaagt tcaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240
atgcagctca acagcctgac atctgaagac tctgcagtct atttctgtgc aagatgggac 300
tataggtacg acgacgggag ggcttactat gttatggact tctggggtca aggaacctca 360
gtcaccgtct cctcaggggg tggaggctct ggtggcggtg gctctggcgg aggtggatcc 420
ggtggcggcg gatctgagct ccagatgacc cagtctccat catctctggc tgcgtctgca 480
ggagaaaagg tcactatgag ctgtaagtcc agtcaaagtg ttttatacag ttcaaatcag 540
aagaactact tggcctggta ccagcagaaa ccagggcagt ctcctaaact gctgatctac 600
tgggcatcca ctagggaatc tggtgtccct gatcgcttca caggcagtgg atctgggaca 660
cattttactc tgaccgtcag cagtgtgcaa gctgaagacc tggcagttta ttactgtcat 720
caatacctct actcgtggac gttcggtgga ggcaccaacc tggaaatcaa acggctcgag 780
cccaaatctt ctgacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgcgggt 840
gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080
aaggcgtacg cgtgcgcggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tccaagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt aactcattat tcaaccaaga agttcaaatt 1500
cccttgaccg aaagttacag cccgaattct agcccaggcc agggcaccca gtctgagaac 1560
agctgcaccc acttcccagg caacctgcct aacatgcttc gagatctccg agatgccttc 1620
agcagagtga agactttctt tcaaatgaag gatcagctgg acaacttgtt gttaaaggag 1680
tccttgctgg aggactttaa gggttacctg ggttgccaag ccttgtctga gatgatccag 1740
ttttacctgg aggaggtgat gccccaagct gagaaccaag acccagacat caaggcgcat 1800
gtgaactccc tgggggagaa cctgaagacc ctcaggctga ggctacggcg ctgtcatcga 1860
tttcttccct gtgaaaacaa gagcaaggcc gtggagcagg tgaagaatgc ctttaataag 1920
ctccaagaga aaggcatcta caaagccatg agtgagtttg acatcttcat caactacata 1980
gaagcctaca tgacaatgaa gatacgaaac taa 2013
<210> 187
<211> 670
<212> PRT
<213> 人工序列
<220>
<223> FUN1::IL10融合蛋白
<400> 187
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr
20 25 30
Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
100 105 110
Asp Phe Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Glu Leu Gln Met Thr Gln Ser Pro Ser Ser Leu Ala Ala Ser Ala
145 150 155 160
Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr
165 170 175
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
195 200 205
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr His Phe Thr Leu
210 215 220
Thr Val Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys His
225 230 235 240
Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile
245 250 255
Lys Arg Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
260 265 270
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
340 345 350
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
355 360 365
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln
485 490 495
Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Ser Pro
500 505 510
Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn
515 520 525
Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys
530 535 540
Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu
545 550 555 560
Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser
565 570 575
Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn
580 585 590
Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu
595 600 605
Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys
610 615 620
Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys
625 630 635 640
Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe
645 650 655
Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
660 665 670
<210> 188
<211> 1983
<212> DNA
<213> 人工序列
<220>
<223> 编码3D1::IL10融合蛋白的核苷酸序列
<400> 188
caggtccagc tgcagcagtc tgggcctgag ctggtgaggc ctggggaatc agtgaagatt 60
tcctgcaagg gttccggcta cacattcact gattatgcta tacagtgggt gaagcagagt 120
catgcaaaga gtctagagtg gattggagtt attaatattt actatgataa tacaaactac 180
aaccagaagt ttaagggcaa ggccacaatg actgtagaca aatcctccag cacagcctat 240
atggaacttg ccagattgac atctgaggat tctgccatct attactgtgc aagagcggcc 300
tggtatatgg actactgggg tcaaggaacc tcagtcaccg tctcctcagg gggtggaggc 360
tctggtggcg gtggctctgg cggaggtgga tccggtggcg gcggatctga cattgtgctg 420
tcacagtctc catcctccct ggctgtgtca gcaggagaga aggtcactat gagctgcaaa 480
tccagtcaga gtctgctcaa cagtagaacc cgagagaact acttggcttg gtaccagcag 540
aaaccagggc agtctcctaa actgctgatc tactgggcat ccactaggga atctggggtc 600
cctgatcgct tcacaggcag tggatctggg acagatttca ctctcaccat cagcagtgtg 660
caggctgaag acctggcagt ttattactgc acgcaatctt ataatcttta cacgttcgga 720
ggggggacca agctggaaat aaaactcgag cccaaatctt ctgacaaaac tcacacatgc 780
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 840
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 900
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 960
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 1020
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 1080
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1140
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 1200
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 1260
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1320
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1380
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1440
aactcattat tcaaccaaga agttcaaatt cccttgaccg aaagttacag cccgaattct 1500
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 1560
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 1620
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 1680
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 1740
gagaaccaag acccagacat caaggcgcat gtgaactccc tgggggagaa cctgaagacc 1800
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 1860
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 1920
agtgagtttg acatcttcat caactacata gaagcctaca tgacaatgaa gatacgaaac 1980
taa 1983
<210> 189
<211> 660
<212> PRT
<213> 人工序列
<220>
<223> 3D1::IL10融合蛋白
<400> 189
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Glu
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile Gln Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile
35 40 45
Gly Val Ile Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Ala Ala Trp Tyr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Ser Gln Ser Pro
130 135 140
Ser Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys
145 150 155 160
Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr Leu Ala
165 170 175
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp
180 185 190
Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
195 200 205
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp
210 215 220
Leu Ala Val Tyr Tyr Cys Thr Gln Ser Tyr Asn Leu Tyr Thr Phe Gly
225 230 235 240
Gly Gly Thr Lys Leu Glu Ile Lys Leu Glu Pro Lys Ser Ser Asp Lys
245 250 255
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
260 265 270
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
275 280 285
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
290 295 300
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
305 310 315 320
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
325 330 335
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala
340 345 350
Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
355 360 365
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
370 375 380
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
385 390 395 400
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
405 410 415
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
420 425 430
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
435 440 445
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
450 455 460
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
465 470 475 480
Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr
485 490 495
Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys
500 505 510
Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp
515 520 525
Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp
530 535 540
Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu
545 550 555 560
Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val
565 570 575
Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn
580 585 590
Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys
595 600 605
His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val
610 615 620
Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met
625 630 635 640
Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met
645 650 655
Lys Ile Arg Asn
660
<210> 190
<211> 1611
<212> DNA
<213> 人工序列
<220>
<223> 编码CTLA4::IL10 I87A融合蛋白的核苷酸序列
<400> 190
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa ctcattattc 1080
aaccaagaag ttcaaattcc cttgaccgaa agttacagcc cgaattctag cccaggccag 1140
ggcacccagt ctgagaacag ctgcacccac ttcccaggca acctgcctaa catgcttcga 1200
gatctccgag atgccttcag cagagtgaag actttctttc aaatgaagga tcagctggac 1260
aacttgttgt taaaggagtc cttgctggag gactttaagg gttacctggg ttgccaagcc 1320
ttgtctgaga tgatccagtt ttacctggag gaggtgatgc cccaagctga gaaccaagac 1380
ccagacgcca aggcgcatgt gaactccctg ggggagaacc tgaagaccct caggctgagg 1440
ctacggcgct gtcatcgatt tcttccctgt gaaaacaaga gcaaggccgt ggagcaggtg 1500
aagaatgcct ttaataagct ccaagagaaa ggcatctaca aagccatgag tgagtttgac 1560
atcttcatca actacataga agcctacatg acaatgaaga tacgaaacta a 1611
<210> 191
<211> 536
<212> PRT
<213> 人工序列
<220>
<223> CTLA4::IL10 I87A融合蛋白
<400> 191
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu
355 360 365
Thr Glu Ser Tyr Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln Ser
370 375 380
Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg
385 390 395 400
Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys
405 410 415
Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe
420 425 430
Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr
435 440 445
Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ala Lys
450 455 460
Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg
465 470 475 480
Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala
485 490 495
Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile
500 505 510
Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala
515 520 525
Tyr Met Thr Met Lys Ile Arg Asn
530 535
<210> 192
<211> 1611
<212> DNA
<213> 人工序列
<220>
<223> 编码CTLA4::IL10 I87S融合蛋白的核苷酸序列
<400> 192
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa ctcattattc 1080
aaccaagaag ttcaaattcc cttgaccgaa agttacagcc cgaattctag cccaggccag 1140
ggcacccagt ctgagaacag ctgcacccac ttcccaggca acctgcctaa catgcttcga 1200
gatctccgag atgccttcag cagagtgaag actttctttc aaatgaagga tcagctggac 1260
aacttgttgt taaaggagtc cttgctggag gactttaagg gttacctggg ttgccaagcc 1320
ttgtctgaga tgatccagtt ttacctggag gaggtgatgc cccaagctga gaaccaagac 1380
ccagacagca aggcgcatgt gaactccctg ggggagaacc tgaagaccct caggctgagg 1440
ctacggcgct gtcatcgatt tcttccctgt gaaaacaaga gcaaggccgt ggagcaggtg 1500
aagaatgcct ttaataagct ccaagagaaa ggcatctaca aagccatgag tgagtttgac 1560
atcttcatca actacataga agcctacatg acaatgaaga tacgaaacta a 1611
<210> 193
<211> 536
<212> PRT
<213> 人工序列
<220>
<223> CTLA4::IL10 I87S融合蛋白
<400> 193
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu
355 360 365
Thr Glu Ser Tyr Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln Ser
370 375 380
Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg
385 390 395 400
Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys
405 410 415
Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe
420 425 430
Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr
435 440 445
Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ser Lys
450 455 460
Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg
465 470 475 480
Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala
485 490 495
Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile
500 505 510
Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala
515 520 525
Tyr Met Thr Met Lys Ile Arg Asn
530 535
<210> 194
<211> 1635
<212> DNA
<213> 人工序列
<220>
<223> 编码含H68接头的小鼠CTLA4::小鼠IL10融合蛋白的核苷酸序列
<400> 194
gccatacagg tgacccaacc ttcagtggtg ttggctagca gccatggtgt cgccagcttt 60
ccatgtgaat attcaccatc acacaacact gatgaggtcc gggtgactgt gctgcggcag 120
acaaatgacc aaatgactga ggtctgtgcc acgacattca cagagaagaa tacagtgggc 180
ttcctagatt accccttctg cagtggtacc tttaatgaaa gcagagtgaa cctcaccatc 240
caaggactga gagctgttga cacgggactg tacctctgca aggtggaact catgtaccca 300
ccgccatact ttgtgggcat gggcaacggg acgcagattt atgtcattga tccagaacca 360
tgcccggatt ctgacctcga gcccagagtg cccataacac agaacccctg tcctccactc 420
aaagagtgtc ccccatgcgc agctccagac gcagcgggtg cgccatccgt cttcatcttc 480
cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 540
gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 600
gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 660
agtgccctcc ccatccagca ccaggactgg atgagtggca aggcgttcgc atgcgcggtc 720
aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 780
agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 840
agtctgacct gcatgatcgc aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 900
aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 960
tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1020
gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1080
tctctgggta aaaactcatt attcaaccaa gaagttcaaa ttcccttgac cgaaagttac 1140
agcccgaatt ctagcagggg ccagtacagc cgggaagaca ataactgcac ccacttccca 1200
gtcggccaga gccacatgct cctagagctg cggactgcct tcagccaggt gaagactttc 1260
tttcaaacaa aggaccagct ggacaacata ctgctaaccg actccttaat gcaggacttt 1320
aagggttact tgggttgcca agccttatcg gaaatgatcc agttttacct ggtagaagtg 1380
atgccccagg cagagaagca tggcccagaa atcaaggagc atttgaactc cctgggtgag 1440
aagctgaaga ccctcaggat gcggctgagg cgctgtcatc gatttctccc ctgtgaaaat 1500
aagagcaagg cagtggagca ggtgaagagt gattttaata agctccaaga ccaaggtgtc 1560
tacaaggcca tgaatgaatt tgacatcttc atcaactgca tagaagcata catgatgatc 1620
aaaatgaaaa gctaa 1635
<210> 195
<211> 544
<212> PRT
<213> 人工序列
<220>
<223> 含H68接头的小鼠CTLA4::小鼠IL10 融合蛋白
<400> 195
Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser His Gly
1 5 10 15
Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His Asn Thr Asp Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln Met Thr Glu Val
35 40 45
Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp Tyr
50 55 60
Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro
115 120 125
Arg Val Pro Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu Cys Pro
130 135 140
Pro Cys Ala Ala Pro Asp Ala Ala Gly Ala Pro Ser Val Phe Ile Phe
145 150 155 160
Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Met Val
165 170 175
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile
180 185 190
Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
195 200 205
His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro
210 215 220
Ile Gln His Gln Asp Trp Met Ser Gly Lys Ala Phe Ala Cys Ala Val
225 230 235 240
Asn Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro
245 250 255
Arg Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Ala
260 265 270
Glu Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys Met Ile Ala Gly
275 280 285
Phe Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn Gly Arg Thr
290 295 300
Glu Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser Asp Gly Ser
305 310 315 320
Tyr Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr Trp Glu Arg
325 330 335
Gly Ser Leu Phe Ala Cys Ser Val Val His Glu Gly Leu His Asn His
340 345 350
Leu Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly Lys Asn Ser Leu Phe
355 360 365
Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser
370 375 380
Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His Phe Pro
385 390 395 400
Val Gly Gln Ser His Met Leu Leu Glu Leu Arg Thr Ala Phe Ser Gln
405 410 415
Val Lys Thr Phe Phe Gln Thr Lys Asp Gln Leu Asp Asn Ile Leu Leu
420 425 430
Thr Asp Ser Leu Met Gln Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
435 440 445
Leu Ser Glu Met Ile Gln Phe Tyr Leu Val Glu Val Met Pro Gln Ala
450 455 460
Glu Lys His Gly Pro Glu Ile Lys Glu His Leu Asn Ser Leu Gly Glu
465 470 475 480
Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys His Arg Phe Leu
485 490 495
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Ser Asp Phe
500 505 510
Asn Lys Leu Gln Asp Gln Gly Val Tyr Lys Ala Met Asn Glu Phe Asp
515 520 525
Ile Phe Ile Asn Cys Ile Glu Ala Tyr Met Met Ile Lys Met Lys Ser
530 535 540
<210> 196
<211> 1638
<212> DNA
<213> 人工序列
<220>
<223> 编码含H75接头的小鼠CTLA4::小鼠IL10融合蛋白的核苷酸序列
<400> 196
gccatacagg tgacccaacc ttcagtggtg ttggctagca gccatggtgt cgccagcttt 60
ccatgtgaat attcaccatc acacaacact gatgaggtcc gggtgactgt gctgcggcag 120
acaaatgacc aaatgactga ggtctgtgcc acgacattca cagagaagaa tacagtgggc 180
ttcctagatt accccttctg cagtggtacc tttaatgaaa gcagagtgaa cctcaccatc 240
caaggactga gagctgttga cacgggactg tacctctgca aggtggaact catgtaccca 300
ccgccatact ttgtgggcat gggcaacggg acgcagattt atgtcattga tccagaacca 360
tgcccggatt ctgacctcga gcccagagtg cccataacac agaacccctg tcctccactc 420
aaagagtgtc ccccatgcgc agctccagac gcagcgggtg cgccatccgt cttcatcttc 480
cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 540
gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 600
gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 660
agtgccctcc ccatccagca ccaggactgg atgagtggca aggcgttcgc atgcgcggtc 720
aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 780
agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 840
agtctgacct gcatgatcac aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 900
aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 960
tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1020
gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1080
tctctgggta aacagaggca caacaattct tccctgaata caggaactca gatggcaggt 1140
cattctccga attctagcag gggccagtac agccgggaag acaataactg cacccacttc 1200
ccagtcggcc agagccacat gctcctagag ctgcggactg ccttcagcca ggtgaagact 1260
ttctttcaaa caaaggacca gctggacaac atactgctaa ccgactcctt aatgcaggac 1320
tttaagggtt acttgggttg ccaagcctta tcggaaatga tccagtttta cctggtagaa 1380
gtgatgcccc aggcagagaa gcatggccca gaaatcaagg agcatttgaa ctccctgggt 1440
gagaagctga agaccctcag gatgcggctg aggcgctgtc atcgatttct cccctgtgaa 1500
aataagagca aggcagtgga gcaggtgaag agtgatttta ataagctcca agaccaaggt 1560
gtctacaagg ccatgaatga atttgacatc ttcatcaact gcatagaagc atacatgatg 1620
atcaaaatga aaagctaa 1638
<210> 197
<211> 545
<212> PRT
<213> 人工序列
<220>
<223> 含H75接头的小鼠CTLA4::小鼠IL10融合蛋白
<400> 197
Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser His Gly
1 5 10 15
Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His Asn Thr Asp Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln Met Thr Glu Val
35 40 45
Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp Tyr
50 55 60
Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro
115 120 125
Arg Val Pro Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu Cys Pro
130 135 140
Pro Cys Ala Ala Pro Asp Ala Ala Gly Ala Pro Ser Val Phe Ile Phe
145 150 155 160
Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Met Val
165 170 175
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile
180 185 190
Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
195 200 205
His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro
210 215 220
Ile Gln His Gln Asp Trp Met Ser Gly Lys Ala Phe Ala Cys Ala Val
225 230 235 240
Asn Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro
245 250 255
Arg Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Ala
260 265 270
Glu Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys Met Ile Thr Gly
275 280 285
Phe Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn Gly Arg Thr
290 295 300
Glu Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser Asp Gly Ser
305 310 315 320
Tyr Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr Trp Glu Arg
325 330 335
Gly Ser Leu Phe Ala Cys Ser Val Val His Glu Gly Leu His Asn His
340 345 350
Leu Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly Lys Gln Arg His Asn
355 360 365
Asn Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Gly His Ser Pro Asn
370 375 380
Ser Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His Phe
385 390 395 400
Pro Val Gly Gln Ser His Met Leu Leu Glu Leu Arg Thr Ala Phe Ser
405 410 415
Gln Val Lys Thr Phe Phe Gln Thr Lys Asp Gln Leu Asp Asn Ile Leu
420 425 430
Leu Thr Asp Ser Leu Met Gln Asp Phe Lys Gly Tyr Leu Gly Cys Gln
435 440 445
Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Val Glu Val Met Pro Gln
450 455 460
Ala Glu Lys His Gly Pro Glu Ile Lys Glu His Leu Asn Ser Leu Gly
465 470 475 480
Glu Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys His Arg Phe
485 490 495
Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Ser Asp
500 505 510
Phe Asn Lys Leu Gln Asp Gln Gly Val Tyr Lys Ala Met Asn Glu Phe
515 520 525
Asp Ile Phe Ile Asn Cys Ile Glu Ala Tyr Met Met Ile Lys Met Lys
530 535 540
Ser
545
<210> 198
<211> 1920
<212> DNA
<213> 人工序列
<220>
<223> 编码GITR::3D1 融合蛋白的核苷酸序列
<400> 198
cagcgcccca ccgggggtcc cgggtgcggc cctgggcgcc tcctgcttgg gacgggaacg 60
gacgcgcgct gctgccgggt tcacacgacg cgctgctgcc gcgattaccc gggcgaggag 120
tgctgttccg agtgggactg catgtgtgtc cagcctgaat tccactgcgg agacccttgc 180
tgcacgacct gccggcacca cccttgtccc ccaggccagg gggtacagtc ccaggggaaa 240
ttcagttttg gcttccagtg tatcgactgt gcctcgggga ccttctccgg gggccacgaa 300
ggccactgca aaccttggac agactgcacc cagttcgggt ttctcactgt gttccctggg 360
aacaagaccc acaacgctgt gtgcgtccca gggtccccgc cggcagagcc gcttgggctc 420
gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 480
ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 540
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 600
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 660
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 720
ggcaaggcgt acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 780
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 840
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 900
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 960
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1020
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1080
tacacgcaga agagcctctc cctgtctccg ggtaactcat tattcaacca agaagttcaa 1140
attcccttga ccgaaagtta cagcccgaat tctcaggtcc agctgcagca gtctgggcct 1200
gagctggtga ggcctgggga atcagtgaag atttcctgca agggttccgg ctacacattc 1260
actgattatg ctatacagtg ggtgaagcag agtcatgcaa agagtctaga gtggattgga 1320
gttattaata tttactatga taatacaaac tacaaccaga agtttaaggg caaggccaca 1380
atgactgtag acaaatcctc cagcacagcc tatatggaac ttgccagatt gacatctgag 1440
gattctgcca tctattactg tgcaagagcg gcctggtata tggactactg gggtcaagga 1500
acctcagtca ccgtctcctc agggggtgga ggctctggtg gcggtggctc tggcggaggt 1560
ggatccggtg gcggcggatc tgacattgtg ctgtcacagt ctccatcctc cctggctgtg 1620
tcagcaggag agaaggtcac tatgagctgc aaatccagtc agagtctgct caacagtaga 1680
acccgagaga actacttggc ttggtaccag cagaaaccag ggcagtctcc taaactgctg 1740
atctactggg catccactag ggaatctggg gtccctgatc gcttcacagg cagtggatct 1800
gggacagatt tcactctcac catcagcagt gtgcaggctg aagacctggc agtttattac 1860
tgcacgcaat cttataatct ttacacgttc ggagggggga ccaagctgga aataaaataa 1920
<210> 199
<211> 639
<212> PRT
<213> 人工序列
<220>
<223> GITR::3D1融合蛋白
<400> 199
Gln Arg Pro Thr Gly Gly Pro Gly Cys Gly Pro Gly Arg Leu Leu Leu
1 5 10 15
Gly Thr Gly Thr Asp Ala Arg Cys Cys Arg Val His Thr Thr Arg Cys
20 25 30
Cys Arg Asp Tyr Pro Gly Glu Glu Cys Cys Ser Glu Trp Asp Cys Met
35 40 45
Cys Val Gln Pro Glu Phe His Cys Gly Asp Pro Cys Cys Thr Thr Cys
50 55 60
Arg His His Pro Cys Pro Pro Gly Gln Gly Val Gln Ser Gln Gly Lys
65 70 75 80
Phe Ser Phe Gly Phe Gln Cys Ile Asp Cys Ala Ser Gly Thr Phe Ser
85 90 95
Gly Gly His Glu Gly His Cys Lys Pro Trp Thr Asp Cys Thr Gln Phe
100 105 110
Gly Phe Leu Thr Val Phe Pro Gly Asn Lys Thr His Asn Ala Val Cys
115 120 125
Val Pro Gly Ser Pro Pro Ala Glu Pro Leu Gly Leu Glu Pro Lys Ser
130 135 140
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
145 150 155 160
Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
165 170 175
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
180 185 190
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
195 200 205
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
210 215 220
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
225 230 235 240
Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro
245 250 255
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
260 265 270
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
275 280 285
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
290 295 300
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
305 310 315 320
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
325 330 335
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
340 345 350
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
355 360 365
Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
370 375 380
Glu Ser Tyr Ser Pro Asn Ser Gln Val Gln Leu Gln Gln Ser Gly Pro
385 390 395 400
Glu Leu Val Arg Pro Gly Glu Ser Val Lys Ile Ser Cys Lys Gly Ser
405 410 415
Gly Tyr Thr Phe Thr Asp Tyr Ala Ile Gln Trp Val Lys Gln Ser His
420 425 430
Ala Lys Ser Leu Glu Trp Ile Gly Val Ile Asn Ile Tyr Tyr Asp Asn
435 440 445
Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp
450 455 460
Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ala Arg Leu Thr Ser Glu
465 470 475 480
Asp Ser Ala Ile Tyr Tyr Cys Ala Arg Ala Ala Trp Tyr Met Asp Tyr
485 490 495
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser
500 505 510
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
515 520 525
Ile Val Leu Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly Glu
530 535 540
Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg
545 550 555 560
Thr Arg Glu Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser
565 570 575
Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro
580 585 590
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
595 600 605
Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Thr Gln Ser
610 615 620
Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
625 630 635
<210> 200
<211> 1989
<212> DNA
<213> 人工序列
<220>
<223> 编码HVEM::3D1 融合蛋白的核苷酸序列
<400> 200
ctgccgtcct gcaaggagga cgagtaccca gtgggctccg agtgctgccc caagtgcagt 60
ccaggttatc gtgtgaagga ggcctgcggg gagctgacgg gcacagtgtg tgaaccctgc 120
cctccaggca cctacattgc ccacctcaat ggcctaagca agtgtctgca gtgccaaatg 180
tgtgacccag ccatgggcct gcgcgcgagc cggaactgct ccaggacaga gaacgccgtg 240
tgtggctgca gcccaggcca cttctgcatc gtccaggacg gggaccactg cgccgcgtgc 300
cgcgcttacg ccacctccag cccgggccag agggtgcaga agggaggcac cgagagtcag 360
gacaccctgt gtcagaactg ccccccgggg accttctctc ccaatgggac cctggaggaa 420
tgtcagcacc agaccaagtg cagctggctg gtgacgaagg ccggagctgg gaccagcagc 480
tcccacctcg agcccaaatc ttctgacaaa actcacacat gcccaccgtg cccagcacct 540
gaagccgcgg gtgcaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 600
atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 660
gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 720
gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 780
tggctgaatg gcaaggcgta cgcgtgcgcg gtctccaaca aagccctccc agcccccatc 840
gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 900
ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 960
tatccaagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1020
accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1080
gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1140
cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaactcatt attcaaccaa 1200
gaagttcaaa ttcccttgac cgaaagttac agcccgaatt ctcaggtcca gctgcagcag 1260
tctgggcctg agctggtgag gcctggggaa tcagtgaaga tttcctgcaa gggttccggc 1320
tacacattca ctgattatgc tatacagtgg gtgaagcaga gtcatgcaaa gagtctagag 1380
tggattggag ttattaatat ttactatgat aatacaaact acaaccagaa gtttaagggc 1440
aaggccacaa tgactgtaga caaatcctcc agcacagcct atatggaact tgccagattg 1500
acatctgagg attctgccat ctattactgt gcaagagcgg cctggtatat ggactactgg 1560
ggtcaaggaa cctcagtcac cgtctcctca gggggtggag gctctggtgg cggtggctct 1620
ggcggaggtg gatccggtgg cggcggatct gacattgtgc tgtcacagtc tccatcctcc 1680
ctggctgtgt cagcaggaga gaaggtcact atgagctgca aatccagtca gagtctgctc 1740
aacagtagaa cccgagagaa ctacttggct tggtaccagc agaaaccagg gcagtctcct 1800
aaactgctga tctactgggc atccactagg gaatctgggg tccctgatcg cttcacaggc 1860
agtggatctg ggacagattt cactctcacc atcagcagtg tgcaggctga agacctggca 1920
gtttattact gcacgcaatc ttataatctt tacacgttcg gaggggggac caagctggaa 1980
ataaaataa 1989
<210> 201
<211> 662
<212> PRT
<213> 人工序列
<220>
<223> HVEM::3D1融合蛋白
<400> 201
Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys
1 5 10 15
Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu
20 25 30
Thr Gly Thr Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His
35 40 45
Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala
50 55 60
Met Gly Leu Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val
65 70 75 80
Cys Gly Cys Ser Pro Gly His Phe Cys Ile Val Gln Asp Gly Asp His
85 90 95
Cys Ala Ala Cys Arg Ala Tyr Ala Thr Ser Ser Pro Gly Gln Arg Val
100 105 110
Gln Lys Gly Gly Thr Glu Ser Gln Asp Thr Leu Cys Gln Asn Cys Pro
115 120 125
Pro Gly Thr Phe Ser Pro Asn Gly Thr Leu Glu Glu Cys Gln His Gln
130 135 140
Thr Lys Cys Ser Trp Leu Val Thr Lys Ala Gly Ala Gly Thr Ser Ser
145 150 155 160
Ser His Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
165 170 175
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
180 185 190
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
195 200 205
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
210 215 220
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
225 230 235 240
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
245 250 255
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
260 265 270
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
275 280 285
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
290 295 300
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
305 310 315 320
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
325 330 335
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
340 345 350
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
355 360 365
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
370 375 380
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln
385 390 395 400
Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Gln Val
405 410 415
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Glu Ser Val
420 425 430
Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala Ile
435 440 445
Gln Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile Gly Val
450 455 460
Ile Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln Lys Phe Lys Gly
465 470 475 480
Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu
485 490 495
Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg
500 505 510
Ala Ala Trp Tyr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
515 520 525
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
530 535 540
Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Ser Gln Ser Pro Ser Ser
545 550 555 560
Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser
565 570 575
Gln Ser Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr Leu Ala Trp Tyr
580 585 590
Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser
595 600 605
Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly
610 615 620
Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala
625 630 635 640
Val Tyr Tyr Cys Thr Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly
645 650 655
Thr Lys Leu Glu Ile Lys
660
<210> 202
<211> 2163
<212> DNA
<213> 人工序列
<220>
<223> 编码PDL1::3D1融合蛋白的核苷酸序列
<400> 202
tttactgtca cggttcccaa ggacctatat gtggtagagt atggtagcaa tatgacaatt 60
gaatgcaaat tcccagtaga aaaacaatta gacctggctg cactaattgt ctattgggaa 120
atggaggata agaacattat tcaatttgtg catggagagg aagacctgaa ggttcagcat 180
agtagctaca gacagagggc ccggctgttg aaggaccagc tctccctggg aaatgctgca 240
cttcagatca cagatgtgaa attgcaggat gcaggggtgt accgctgcat gatcagctat 300
ggtggtgccg actacaagcg aattactgtg aaagtcaatg ccccatacaa caaaatcaac 360
caaagaattt tggttgtgga tccagtcacc tctgaacatg aactgacatg tcaggctgag 420
ggctacccca aggccgaagt catctggaca agcagtgacc atcaagtcct gagtggtaag 480
accaccacca ccaattccaa gagagaggag aagcttttca atgtgaccag cacactgaga 540
atcaacacaa caactaatga gattttctac tgcactttta ggagattaga tcctgaggaa 600
aaccatacag ctgaattggt catcccagaa ctacctctgg cacatcctcc aaatgaaagg 660
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 720
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaact cattattcaa ccaagaagtt 1380
caaattccct tgaccgaaag ttacagcccg aattctcagg tccagctgca gcagtctggg 1440
cctgagctgg tgaggcctgg ggaatcagtg aagatttcct gcaagggttc cggctacaca 1500
ttcactgatt atgctataca gtgggtgaag cagagtcatg caaagagtct agagtggatt 1560
ggagttatta atatttacta tgataataca aactacaacc agaagtttaa gggcaaggcc 1620
acaatgactg tagacaaatc ctccagcaca gcctatatgg aacttgccag attgacatct 1680
gaggattctg ccatctatta ctgtgcaaga gcggcctggt atatggacta ctggggtcaa 1740
ggaacctcag tcaccgtctc ctcagggggt ggaggctctg gtggcggtgg ctctggcgga 1800
ggtggatccg gtggcggcgg atctgacatt gtgctgtcac agtctccatc ctccctggct 1860
gtgtcagcag gagagaaggt cactatgagc tgcaaatcca gtcagagtct gctcaacagt 1920
agaacccgag agaactactt ggcttggtac cagcagaaac cagggcagtc tcctaaactg 1980
ctgatctact gggcatccac tagggaatct ggggtccctg atcgcttcac aggcagtgga 2040
tctgggacag atttcactct caccatcagc agtgtgcagg ctgaagacct ggcagtttat 2100
tactgcacgc aatcttataa tctttacacg ttcggagggg ggaccaagct ggaaataaaa 2160
taa 2163
<210> 203
<211> 720
<212> PRT
<213> 人工序列
<220>
<223> PDL1::3D1融合蛋白
<400> 203
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Leu Glu Pro Lys
210 215 220
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
225 230 235 240
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
305 310 315 320
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
355 360 365
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
435 440 445
Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu
450 455 460
Thr Glu Ser Tyr Ser Pro Asn Ser Gln Val Gln Leu Gln Gln Ser Gly
465 470 475 480
Pro Glu Leu Val Arg Pro Gly Glu Ser Val Lys Ile Ser Cys Lys Gly
485 490 495
Ser Gly Tyr Thr Phe Thr Asp Tyr Ala Ile Gln Trp Val Lys Gln Ser
500 505 510
His Ala Lys Ser Leu Glu Trp Ile Gly Val Ile Asn Ile Tyr Tyr Asp
515 520 525
Asn Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val
530 535 540
Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ala Arg Leu Thr Ser
545 550 555 560
Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg Ala Ala Trp Tyr Met Asp
565 570 575
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 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
Asp Ile Val Leu Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly
610 615 620
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
625 630 635 640
Arg Thr Arg Glu Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
645 650 655
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
660 665 670
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
675 680 685
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Thr Gln
690 695 700
Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
705 710 715 720
<210> 204
<211> 2106
<212> DNA
<213> 人工序列
<220>
<223> 编码PDL2::3D1融合蛋白的核苷酸序列
<400> 204
ttattcacag tgacagtccc taaggaactg tacataatag agcatggcag caatgtgacc 60
ctggaatgca actttgacac tggaagtcat gtgaaccttg gagcaataac agccagtttg 120
caaaaggtgg aaaatgatac atccccacac cgtgaaagag ccactttgct ggaggagcag 180
ctgcccttag ggaaggcctc gttccacata cctcaagtcc aagtgaggga cgaaggacag 240
taccaatgca taatcatcta tggggtcgcc tgggactaca agtacctgac tctgaaagtc 300
aaagcttcct acaggaaaat aaacactcac atcctaaagg ttccagaaac agatgaggta 360
gagctcacct gccaggctac aggttatcct ctggcagaag tatcctggcc aaacgtcagc 420
gttcctgcca acaccagcca ctccaggacc cctgaaggcc tctaccaggt caccagtgtt 480
ctgcgcctaa agccaccccc tggcagaaac ttcagctgtg tgttctggaa tactcacgtg 540
agggaactta ctttggccag cattgacctt caaagtcaga tggaacccag gacccatcca 600
actctcgagc ccaaatcttc tgacaaaact cacacatgcc caccgtgccc agcacctgaa 660
gccgcgggtg caccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 720
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 780
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 840
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 900
ctgaatggca aggcgtacgc gtgcgcggtc tccaacaaag ccctcccagc ccccatcgag 960
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1020
tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1080
ccaagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1140
acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1200
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1260
aaccactaca cgcagaagag cctctccctg tctccgggta actcattatt caaccaagaa 1320
gttcaaattc ccttgaccga aagttacagc ccgaattctc aggtccagct gcagcagtct 1380
gggcctgagc tggtgaggcc tggggaatca gtgaagattt cctgcaaggg ttccggctac 1440
acattcactg attatgctat acagtgggtg aagcagagtc atgcaaagag tctagagtgg 1500
attggagtta ttaatattta ctatgataat acaaactaca accagaagtt taagggcaag 1560
gccacaatga ctgtagacaa atcctccagc acagcctata tggaacttgc cagattgaca 1620
tctgaggatt ctgccatcta ttactgtgca agagcggcct ggtatatgga ctactggggt 1680
caaggaacct cagtcaccgt ctcctcaggg ggtggaggct ctggtggcgg tggctctggc 1740
ggaggtggat ccggtggcgg cggatctgac attgtgctgt cacagtctcc atcctccctg 1800
gctgtgtcag caggagagaa ggtcactatg agctgcaaat ccagtcagag tctgctcaac 1860
agtagaaccc gagagaacta cttggcttgg taccagcaga aaccagggca gtctcctaaa 1920
ctgctgatct actgggcatc cactagggaa tctggggtcc ctgatcgctt cacaggcagt 1980
ggatctggga cagatttcac tctcaccatc agcagtgtgc aggctgaaga cctggcagtt 2040
tattactgca cgcaatctta taatctttac acgttcggag gggggaccaa gctggaaata 2100
aaataa 2106
<210> 205
<211> 701
<212> PRT
<213> 人工序列
<220>
<223> PDL2::3D1融合蛋白
<400> 205
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Leu Glu Pro Lys Ser Ser Asp
195 200 205
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala
210 215 220
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
225 230 235 240
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
245 250 255
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
260 265 270
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
275 280 285
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
290 295 300
Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
305 310 315 320
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
325 330 335
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
340 345 350
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
355 360 365
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
370 375 380
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
385 390 395 400
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
405 410 415
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
420 425 430
Gly Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser
435 440 445
Tyr Ser Pro Asn Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
450 455 460
Val Arg Pro Gly Glu Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr
465 470 475 480
Thr Phe Thr Asp Tyr Ala Ile Gln Trp Val Lys Gln Ser His Ala Lys
485 490 495
Ser Leu Glu Trp Ile Gly Val Ile Asn Ile Tyr Tyr Asp Asn Thr Asn
500 505 510
Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser
515 520 525
Ser Ser Thr Ala Tyr Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser
530 535 540
Ala Ile Tyr Tyr Cys Ala Arg Ala Ala Trp Tyr Met Asp Tyr Trp Gly
545 550 555 560
Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
565 570 575
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val
580 585 590
Leu Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly Glu Lys Val
595 600 605
Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg
610 615 620
Glu Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys
625 630 635 640
Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg
645 650 655
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
660 665 670
Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Thr Gln Ser Tyr Asn
675 680 685
Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
690 695 700
<210> 206
<211> 1806
<212> DNA
<213> 人工序列
<220>
<223> 编码TGFβR1::3D1融合蛋白的核苷酸序列
<400> 206
gcggcgctgc tcccgggggc gacggcgtta cagtgtttct gccacctctg tacaaaagac 60
aattttactt gtgtgacaga tgggctctgc tttgtctctg tcacagagac cacagacaaa 120
gttatacaca acagcatgtg tatagctgaa attgacttaa ttccgcgaga taggccgttt 180
gtatgtgcac cctcttcaaa aactgggtct gtgactacaa catattgctg caatcaggac 240
cattgcaata aaatagaact tccaactact gtaaagtcat cacctggcct tggtcctgtg 300
gaactcgagc ccaaatcttc tgacaaaact cacacatgcc caccgtgccc agcacctgaa 360
gccgcgggtg caccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 420
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 480
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 540
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 600
ctgaatggca aggcgtacgc gtgcgcggtc tccaacaaag ccctcccagc ccccatcgag 660
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 720
tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 780
ccaagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 840
acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 900
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 960
aaccactaca cgcagaagag cctctccctg tctccgggta actcattatt caaccaagaa 1020
gttcaaattc ccttgaccga aagttacagc ccgaattctc aggtccagct gcagcagtct 1080
gggcctgagc tggtgaggcc tggggaatca gtgaagattt cctgcaaggg ttccggctac 1140
acattcactg attatgctat acagtgggtg aagcagagtc atgcaaagag tctagagtgg 1200
attggagtta ttaatattta ctatgataat acaaactaca accagaagtt taagggcaag 1260
gccacaatga ctgtagacaa atcctccagc acagcctata tggaacttgc cagattgaca 1320
tctgaggatt ctgccatcta ttactgtgca agagcggcct ggtatatgga ctactggggt 1380
caaggaacct cagtcaccgt ctcctcaggg ggtggaggct ctggtggcgg tggctctggc 1440
ggaggtggat ccggtggcgg cggatctgac attgtgctgt cacagtctcc atcctccctg 1500
gctgtgtcag caggagagaa ggtcactatg agctgcaaat ccagtcagag tctgctcaac 1560
agtagaaccc gagagaacta cttggcttgg taccagcaga aaccagggca gtctcctaaa 1620
ctgctgatct actgggcatc cactagggaa tctggggtcc ctgatcgctt cacaggcagt 1680
ggatctggga cagatttcac tctcaccatc agcagtgtgc aggctgaaga cctggcagtt 1740
tattactgca cgcaatctta taatctttac acgttcggag gggggaccaa gctggaaata 1800
aaataa 1806
<210> 207
<211> 601
<212> PRT
<213> 人工序列
<220>
<223> TGFβR1::3D1融合蛋白
<400> 207
Ala Ala Leu Leu Pro Gly Ala Thr Ala Leu Gln Cys Phe Cys His Leu
1 5 10 15
Cys Thr Lys Asp Asn Phe Thr Cys Val Thr Asp Gly Leu Cys Phe Val
20 25 30
Ser Val Thr Glu Thr Thr Asp Lys Val Ile His Asn Ser Met Cys Ile
35 40 45
Ala Glu Ile Asp Leu Ile Pro Arg Asp Arg Pro Phe Val Cys Ala Pro
50 55 60
Ser Ser Lys Thr Gly Ser Val Thr Thr Thr Tyr Cys Cys Asn Gln Asp
65 70 75 80
His Cys Asn Lys Ile Glu Leu Pro Thr Thr Val Lys Ser Ser Pro Gly
85 90 95
Leu Gly Pro Val Glu Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr
100 105 110
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe
115 120 125
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
130 135 140
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
145 150 155 160
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
165 170 175
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
180 185 190
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys
195 200 205
Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
210 215 220
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
225 230 235 240
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
245 250 255
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
260 265 270
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
275 280 285
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
290 295 300
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
305 310 315 320
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu
325 330 335
Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn
340 345 350
Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly
355 360 365
Glu Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp
370 375 380
Tyr Ala Ile Gln Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp
385 390 395 400
Ile Gly Val Ile Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln Lys
405 410 415
Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala
420 425 430
Tyr Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr
435 440 445
Cys Ala Arg Ala Ala Trp Tyr Met Asp Tyr Trp Gly Gln Gly Thr Ser
450 455 460
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
465 470 475 480
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Ser Gln Ser
485 490 495
Pro Ser Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys
500 505 510
Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr Leu
515 520 525
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
530 535 540
Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser
545 550 555 560
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu
565 570 575
Asp Leu Ala Val Tyr Tyr Cys Thr Gln Ser Tyr Asn Leu Tyr Thr Phe
580 585 590
Gly Gly Gly Thr Lys Leu Glu Ile Lys
595 600
<210> 208
<211> 1629
<212> DNA
<213> 人工序列
<220>
<223> 编码CTLA4::单IL10 I87A融合蛋白的核苷酸序列
<400> 208
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa ctcattattc 1080
aaccaagaag ttcaaattcc cttgaccgaa agttacagcc cgaattctag cccaggccag 1140
ggcacccagt ctgagaacag ctgcacccac ttcccaggca acctgcctaa catgcttcga 1200
gatctccgag atgccttcag cagagtgaag actttctttc aaatgaagga tcagctggac 1260
aacttgttgt taaaggagtc cttgctggag gactttaagg gttacctggg ttgccaagcc 1320
ttgtctgaga tgatccagtt ttacctggag gaggtgatgc cccaagctga gaaccaagac 1380
ccagacgcca aggcgcatgt gaactccctg ggggagaacc tgaagaccct caggctgagg 1440
ctacggcgct gtcatcgatt tcttccctgt gaaaacggtg gtggatccgg cggtaagagc 1500
aaggccgtgg agcaggtgaa gaatgccttt aataagctcc aagagaaagg catctacaaa 1560
gccatgagtg agtttgacat cttcatcaac tacatagaag cctacatgac aatgaagata 1620
cgaaactaa 1629
<210> 209
<211> 542
<212> PRT
<213> 人工序列
<220>
<223> CTLA4::单IL10 I87A融合蛋白
<400> 209
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu
355 360 365
Thr Glu Ser Tyr Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln Ser
370 375 380
Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg
385 390 395 400
Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys
405 410 415
Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe
420 425 430
Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr
435 440 445
Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ala Lys
450 455 460
Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg
465 470 475 480
Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser
485 490 495
Gly Gly Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys
500 505 510
Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe
515 520 525
Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
530 535 540
<210> 210
<211> 2001
<212> DNA
<213> 人工序列
<220>
<223> 编码单IL10::3D1融合蛋白的核苷酸序列
<400> 210
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacat caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacgg tggtggatcc 360
ggcggtaaga gcaaggccgt ggagcaggtg aagaatgcct ttaataagct ccaagagaaa 420
ggcatctaca aagccatgag tgagtttgac atcttcatca actacataga agcctacatt 480
acaatgaaga tacgaaacct cgagcccaaa tcttctgaca aaactcacac atgcccaccg 540
tgcccagcac ctgaagccgc gggtgcaccg tcagtcttcc tcttcccccc aaaacccaag 600
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 660
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 720
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 780
ctgcaccagg actggctgaa tggcaaggcg tacgcgtgcg cggtctccaa caaagccctc 840
ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 900
tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 960
gtcaaaggct tctatccaag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1020
aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1080
aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1140
catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaactca 1200
ttattcaacc aagaagttca aattcccttg accgaaagtt acagcccgaa ttctcaggtc 1260
cagctgcagc agtctgggcc tgagctggtg aggcctgggg aatcagtgaa gatttcctgc 1320
aagggttccg gctacacatt cactgattat gctatacagt gggtgaagca gagtcatgca 1380
aagagtctag agtggattgg agttattaat atttactatg ataatacaaa ctacaaccag 1440
aagtttaagg gcaaggccac aatgactgta gacaaatcct ccagcacagc ctatatggaa 1500
cttgccagat tgacatctga ggattctgcc atctattact gtgcaagagc ggcctggtat 1560
atggactact ggggtcaagg aacctcagtc accgtctcct cagggggtgg aggctctggt 1620
ggcggtggct ctggcggagg tggatccggt ggcggcggat ctgacattgt gctgtcacag 1680
tctccatcct ccctggctgt gtcagcagga gagaaggtca ctatgagctg caaatccagt 1740
cagagtctgc tcaacagtag aacccgagag aactacttgg cttggtacca gcagaaacca 1800
gggcagtctc ctaaactgct gatctactgg gcatccacta gggaatctgg ggtccctgat 1860
cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcag tgtgcaggct 1920
gaagacctgg cagtttatta ctgcacgcaa tcttataatc tttacacgtt cggagggggg 1980
accaagctgg aaataaaata a 2001
<210> 211
<211> 666
<212> PRT
<213> 人工序列
<220>
<223> 单IL10::3D1融合蛋白
<400> 211
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu
115 120 125
Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
130 135 140
Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile
145 150 155 160
Thr Met Lys Ile Arg Asn Leu Glu Pro Lys Ser Ser Asp Lys Thr His
165 170 175
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val
180 185 190
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
195 200 205
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
210 215 220
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
225 230 235 240
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
245 250 255
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala
260 265 270
Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
275 280 285
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
290 295 300
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
305 310 315 320
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
325 330 335
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
340 345 350
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
355 360 365
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
370 375 380
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Asn Ser
385 390 395 400
Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro
405 410 415
Asn Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro
420 425 430
Gly Glu Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr
435 440 445
Asp Tyr Ala Ile Gln Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu
450 455 460
Trp Ile Gly Val Ile Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln
465 470 475 480
Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr
485 490 495
Ala Tyr Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr
500 505 510
Tyr Cys Ala Arg Ala Ala Trp Tyr Met Asp Tyr Trp Gly Gln Gly Thr
515 520 525
Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
530 535 540
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Ser Gln
545 550 555 560
Ser Pro Ser Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser
565 570 575
Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr
580 585 590
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
595 600 605
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly
610 615 620
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala
625 630 635 640
Glu Asp Leu Ala Val Tyr Tyr Cys Thr Gln Ser Tyr Asn Leu Tyr Thr
645 650 655
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
660 665
<210> 212
<211> 1983
<212> DNA
<213> 人工序列
<220>
<223> 编码IL10 I87A::3D1融合蛋白的核苷酸序列
<400> 212
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacgc caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggtaact cattattcaa ccaagaagtt 1200
caaattccct tgaccgaaag ttacagcccg aattctcagg tccagctgca gcagtctggg 1260
cctgagctgg tgaggcctgg ggaatcagtg aagatttcct gcaagggttc cggctacaca 1320
ttcactgatt atgctataca gtgggtgaag cagagtcatg caaagagtct agagtggatt 1380
ggagttatta atatttacta tgataataca aactacaacc agaagtttaa gggcaaggcc 1440
acaatgactg tagacaaatc ctccagcaca gcctatatgg aacttgccag attgacatct 1500
gaggattctg ccatctatta ctgtgcaaga gcggcctggt atatggacta ctggggtcaa 1560
ggaacctcag tcaccgtctc ctcagggggt ggaggctctg gtggcggtgg ctctggcgga 1620
ggtggatccg gtggcggcgg atctgacatt gtgctgtcac agtctccatc ctccctggct 1680
gtgtcagcag gagagaaggt cactatgagc tgcaaatcca gtcagagtct gctcaacagt 1740
agaacccgag agaactactt ggcttggtac cagcagaaac cagggcagtc tcctaaactg 1800
ctgatctact gggcatccac tagggaatct ggggtccctg atcgcttcac aggcagtgga 1860
tctgggacag atttcactct caccatcagc agtgtgcagg ctgaagacct ggcagtttat 1920
tactgcacgc aatcttataa tctttacacg ttcggagggg ggaccaagct ggaaataaaa 1980
taa 1983
<210> 213
<211> 660
<212> PRT
<213> 人工序列
<220>
<223> IL10 I87A::3D1融合蛋白
<400> 213
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val
385 390 395 400
Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Gln Val Gln Leu
405 410 415
Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Glu Ser Val Lys Ile
420 425 430
Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala Ile Gln Trp
435 440 445
Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile Gly Val Ile Asn
450 455 460
Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala
465 470 475 480
Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ala
485 490 495
Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg Ala Ala
500 505 510
Trp Tyr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Asp Ile Val Leu Ser Gln Ser Pro Ser Ser Leu Ala
545 550 555 560
Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser
565 570 575
Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr Leu Ala Trp Tyr Gln Gln
580 585 590
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
595 600 605
Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
610 615 620
Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr
625 630 635 640
Tyr Cys Thr Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys
645 650 655
Leu Glu Ile Lys
660
<210> 214
<211> 2001
<212> DNA
<213> 人工序列
<220>
<223> 编码单IL10 I87A::3D1融合蛋白的核苷酸序列
<400> 214
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacgc caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacgg tggtggatcc 360
ggcggtaaga gcaaggccgt ggagcaggtg aagaatgcct ttaataagct ccaagagaaa 420
ggcatctaca aagccatgag tgagtttgac atcttcatca actacataga agcctacatt 480
acaatgaaga tacgaaacct cgagcccaaa tcttctgaca aaactcacac atgcccaccg 540
tgcccagcac ctgaagccgc gggtgcaccg tcagtcttcc tcttcccccc aaaacccaag 600
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 660
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 720
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 780
ctgcaccagg actggctgaa tggcaaggcg tacgcgtgcg cggtctccaa caaagccctc 840
ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 900
tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 960
gtcaaaggct tctatccaag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1020
aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1080
aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1140
catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaactca 1200
ttattcaacc aagaagttca aattcccttg accgaaagtt acagcccgaa ttctcaggtc 1260
cagctgcagc agtctgggcc tgagctggtg aggcctgggg aatcagtgaa gatttcctgc 1320
aagggttccg gctacacatt cactgattat gctatacagt gggtgaagca gagtcatgca 1380
aagagtctag agtggattgg agttattaat atttactatg ataatacaaa ctacaaccag 1440
aagtttaagg gcaaggccac aatgactgta gacaaatcct ccagcacagc ctatatggaa 1500
cttgccagat tgacatctga ggattctgcc atctattact gtgcaagagc ggcctggtat 1560
atggactact ggggtcaagg aacctcagtc accgtctcct cagggggtgg aggctctggt 1620
ggcggtggct ctggcggagg tggatccggt ggcggcggat ctgacattgt gctgtcacag 1680
tctccatcct ccctggctgt gtcagcagga gagaaggtca ctatgagctg caaatccagt 1740
cagagtctgc tcaacagtag aacccgagag aactacttgg cttggtacca gcagaaacca 1800
gggcagtctc ctaaactgct gatctactgg gcatccacta gggaatctgg ggtccctgat 1860
cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcag tgtgcaggct 1920
gaagacctgg cagtttatta ctgcacgcaa tcttataatc tttacacgtt cggagggggg 1980
accaagctgg aaataaaata a 2001
<210> 215
<211> 666
<212> PRT
<213> 人工序列
<220>
<223> 单IL10 I87A::3D1融合蛋白
<400> 215
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu
115 120 125
Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
130 135 140
Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile
145 150 155 160
Thr Met Lys Ile Arg Asn Leu Glu Pro Lys Ser Ser Asp Lys Thr His
165 170 175
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val
180 185 190
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
195 200 205
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
210 215 220
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
225 230 235 240
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
245 250 255
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala
260 265 270
Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
275 280 285
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
290 295 300
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
305 310 315 320
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
325 330 335
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
340 345 350
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
355 360 365
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
370 375 380
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Asn Ser
385 390 395 400
Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro
405 410 415
Asn Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro
420 425 430
Gly Glu Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr
435 440 445
Asp Tyr Ala Ile Gln Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu
450 455 460
Trp Ile Gly Val Ile Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln
465 470 475 480
Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr
485 490 495
Ala Tyr Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr
500 505 510
Tyr Cys Ala Arg Ala Ala Trp Tyr Met Asp Tyr Trp Gly Gln Gly Thr
515 520 525
Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
530 535 540
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Ser Gln
545 550 555 560
Ser Pro Ser Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser
565 570 575
Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr
580 585 590
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
595 600 605
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly
610 615 620
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala
625 630 635 640
Glu Asp Leu Ala Val Tyr Tyr Cys Thr Gln Ser Tyr Asn Leu Tyr Thr
645 650 655
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
660 665
<210> 216
<211> 1071
<212> DNA
<213> 人工序列
<220>
<223> LEA29-Fc - CTLA4变体
<400> 216
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaatacact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacg agggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagccggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtta a 1071
<210> 217
<211> 356
<212> PRT
<213> 人工序列
<220>
<223> LEA29-Fc - CTLA4变体
<400> 217
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly
355
<210> 218
<211> 1614
<212> DNA
<213> 人工序列
<220>
<223> 编码含H75接头的LEA29Y::IL10融合蛋白的核苷酸序列
<400> 218
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaatacact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacg agggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtca gaggcacaac 1080
aattcttccc tgaatacagg aactcagatg gcaggtcatt ctccgaattc tagcccaggc 1140
cagggcaccc agtctgagaa cagctgcacc cacttcccag gcaacctgcc taacatgctt 1200
cgagatctcc gagatgcctt cagcagagtg aagactttct ttcaaatgaa ggatcagctg 1260
gacaacttgt tgttaaagga gtccttgctg gaggacttta agggttacct gggttgccaa 1320
gccttgtctg agatgatcca gttttacctg gaggaggtga tgccccaagc tgagaaccaa 1380
gacccagacg ccaaggcgca tgtgaactcc ctgggggaga acctgaagac cctcaggctg 1440
aggctacggc gctgtcatcg atttcttccc tgtgaaaaca agagcaaggc cgtggagcag 1500
gtgaagaatg cctttaataa gctccaagag aaaggcatct acaaagccat gagtgagttt 1560
gacatcttca tcaactacat agaagcctac atgacaatga agatacgaaa ctaa 1614
<210> 219
<211> 537
<212> PRT
<213> 人工序列
<220>
<223> 含H75接头的LEA29Y::IL10融合蛋白
<400> 219
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Gly Thr
355 360 365
Gln Met Ala Gly His Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln
370 375 380
Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu
385 390 395 400
Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met
405 410 415
Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp
420 425 430
Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe
435 440 445
Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ala
450 455 460
Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu
465 470 475 480
Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys
485 490 495
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly
500 505 510
Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu
515 520 525
Ala Tyr Met Thr Met Lys Ile Arg Asn
530 535
<210> 220
<211> 1602
<212> DNA
<213> 人工序列
<220>
<223> 编码含H79接头的CTLA4::单IL10融合蛋白的核苷酸序列
<400> 220
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa ctcattattc 1080
aaccaagaag ttccgaattc tagcccaggc cagggcaccc agtctgagaa cagctgcacc 1140
cacttcccag gcaacctgcc taacatgctt cgagatctcc gagatgcctt cagcagagtg 1200
aagactttct ttcaaatgaa ggatcagctg gacaacttgt tgttaaagga gtccttgctg 1260
gaggacttta agggttacct gggttgccaa gccttgtctg agatgatcca gttttacctg 1320
gaggaggtga tgccccaagc tgagaaccaa gacccagaca tcaaggcgca tgtgaactcc 1380
ctgggggaga acctgaagac cctcaggctg aggctacggc gctgtcatcg atttcttccc 1440
tgtgaaaacg gtggtggatc cggcggtaag agcaaggccg tggagcaggt gaagaatgcc 1500
tttaataagc tccaagagaa aggcatctac aaagccatga gtgagtttga catcttcatc 1560
aactacatag aagcctacat gacaatgaag atacgaaact aa 1602
<210> 221
<211> 533
<212> PRT
<213> 人工序列
<220>
<223> 含H79接头的CTLA4::单IL10融合蛋白
<400> 221
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Pro Asn Ser Ser
355 360 365
Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly
370 375 380
Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val
385 390 395 400
Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys
405 410 415
Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu
420 425 430
Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu
435 440 445
Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn
450 455 460
Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro
465 470 475 480
Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln
485 490 495
Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala
500 505 510
Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr
515 520 525
Met Lys Ile Arg Asn
530
<210> 222
<211> 1608
<212> DNA
<213> 人工序列
<220>
<223> 编码含H80接头的CTLA4::单IL10融合蛋白的核苷酸序列
<400> 222
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtca gaggcacaac 1080
aattcttccc tgaatacacc gaattctagc ccaggccagg gcacccagtc tgagaacagc 1140
tgcacccact tcccaggcaa cctgcctaac atgcttcgag atctccgaga tgccttcagc 1200
agagtgaaga ctttctttca aatgaaggat cagctggaca acttgttgtt aaaggagtcc 1260
ttgctggagg actttaaggg ttacctgggt tgccaagcct tgtctgagat gatccagttt 1320
tacctggagg aggtgatgcc ccaagctgag aaccaagacc cagacatcaa ggcgcatgtg 1380
aactccctgg gggagaacct gaagaccctc aggctgaggc tacggcgctg tcatcgattt 1440
cttccctgtg aaaacggtgg tggatccggc ggtaagagca aggccgtgga gcaggtgaag 1500
aatgccttta ataagctcca agagaaaggc atctacaaag ccatgagtga gtttgacatc 1560
ttcatcaact acatagaagc ctacatgaca atgaagatac gaaactaa 1608
<210> 223
<211> 535
<212> PRT
<213> 人工序列
<220>
<223> 含H80接头的CTLA4::单IL10融合蛋白
<400> 223
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Pro Asn
355 360 365
Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe
370 375 380
Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser
385 390 395 400
Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu
405 410 415
Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln
420 425 430
Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln
435 440 445
Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly
450 455 460
Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe
465 470 475 480
Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val
485 490 495
Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr
500 505 510
Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr
515 520 525
Met Thr Met Lys Ile Arg Asn
530 535
<210> 224
<211> 1473
<212> DNA
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0114 – 产生的人源化FUN1变体
<400> 224
gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggctac agtgaaaatc 60
tcctgcaagg tttctggata caccttcacc gactacaaca tgaactgggt gcaacaggcc 120
cctggaaaag ggcttgagtg gatgggaaat attgatcctt actatggtgg tactagttac 180
aatcagaagt tcaagggcag agtcaccata accgcggaca cgtctacaga cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aacatgggac 300
tataggtacg acgacgggag ggcttactat gttatggact tctggggcca agggaccacg 360
gtcaccgtct cctcaggtgg aggcggttca ggcggaggtg gatccggcgg tggcggatcg 420
ggtggcggcg gatctgacat cgtgatgacc cagtctccag actccctggc tgtgtctctg 480
ggcgagaggg ccaccatcaa ctgcaagtcc agccagagtg ttttatacag ctccaaccag 540
aagaactact tagcttggta ccagcagaaa ccaggacagc ctcctaagct gctcatttac 600
tgggcatcta cccgggaatc cggggtccct gaccgattca gtggcagcgg gtctgggaca 660
gatttcactc tcaccatcag cagcctgcag gctgaagatg tggcagttta ttactgtcat 720
caatacctct actcgtggac gtttggccag gggaccaagc tggagatcaa acggctcgag 780
cccaaatctt ctgacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgcgggt 840
gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080
aaggcgtacg cgtgcgcggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tccaagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagccgg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt taa 1473
<210> 225
<211> 490
<212> PRT
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0114 – 产生的人源化FUN1变体
<400> 225
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
100 105 110
Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
145 150 155 160
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
165 170 175
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
195 200 205
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
210 215 220
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His
225 230 235 240
Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
245 250 255
Lys Arg Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
260 265 270
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
340 345 350
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
355 360 365
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
485 490
<210> 226
<211> 1473
<212> DNA
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0115 – 产生的人源化FUN1变体
<400> 226
caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60
tcctgcaagg cttctggata caccttcacc gactacaaca tgaactgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaaat attgatcctt actatggtgg tactagttac 180
aatcagaagt tcaagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240
atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagatgggac 300
tataggtacg acgacgggag ggcttactat gttatggact tctggggcca agggaccacg 360
gtcaccgtct cctcaggtgg aggcggttca ggcggaggtg gatccggcgg tggcggatcg 420
ggtggcggcg gatctgacat cgtgatgacc cagtctccag actccctggc tgtgtctctg 480
ggcgagaggg ccaccatcaa ctgcaagtcc agccagagtg ttttatacag ctccaaccag 540
aagaactact tagcttggta ccagcagaaa ccaggacagc ctcctaagct gctcatttac 600
tgggcatcta cccgggaatc cggggtccct gaccgattca gtggcagcgg gtctgggaca 660
gatttcactc tcaccatcag cagcctgcag gctgaagatg tggcagttta ttactgtcat 720
caatacctct actcgtggac gtttggccag gggaccaagc tggagatcaa acggctcgag 780
cccaaatctt ctgacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgcgggt 840
gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080
aaggcgtacg cgtgcgcggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tccaagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagccgg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt taa 1473
<210> 227
<211> 490
<212> PRT
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0115 – 产生的人源化FUN1变体
<400> 227
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
100 105 110
Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
145 150 155 160
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
165 170 175
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
195 200 205
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
210 215 220
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His
225 230 235 240
Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
245 250 255
Lys Arg Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
260 265 270
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
340 345 350
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
355 360 365
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
485 490
<210> 228
<211> 1473
<212> DNA
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0116 – 产生的人源化FUN1变体
<400> 228
caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt gactacaaca tgaactggat acgccaggct 120
ccagggaagg ggctggagtg ggtttcaaat attgatcctt actatggtgg tactagttac 180
aatcagaagt tcaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagatgggac 300
tataggtacg acgacgggag ggcttactat gttatggact tctggggcca agggaccacg 360
gtcaccgtct cctcaggtgg aggcggttca ggcggaggtg gatccggcgg tggcggatcg 420
ggtggcggcg gatctgacat cgtgatgacc cagtctccag actccctggc tgtgtctctg 480
ggcgagaggg ccaccatcaa ctgcaagtcc agccagagtg ttttatacag ctccaaccag 540
aagaactact tagcttggta ccagcagaaa ccaggacagc ctcctaagct gctcatttac 600
tgggcatcta cccgggaatc cggggtccct gaccgattca gtggcagcgg gtctgggaca 660
gatttcactc tcaccatcag cagcctgcag gctgaagatg tggcagttta ttactgtcat 720
caatacctct actcgtggac gtttggccag gggaccaagc tggagatcaa acggctcgag 780
cccaaatctt ctgacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgcgggt 840
gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080
aaggcgtacg cgtgcgcggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tccaagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagccgg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt taa 1473
<210> 229
<211> 490
<212> PRT
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0116 – 产生的人源化FUN1变体
<400> 229
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Asn Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
100 105 110
Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
145 150 155 160
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
165 170 175
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
195 200 205
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
210 215 220
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His
225 230 235 240
Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
245 250 255
Lys Arg Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
260 265 270
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
340 345 350
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
355 360 365
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
485 490
<210> 230
<211> 1473
<212> DNA
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0117 – 产生的人源化FUN1变体
<400> 230
gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggctac agtgaaaatc 60
tcctgcaagg tttctggata caccttcacc gactacaaca tgaactgggt gcaacaggcc 120
cctggaaaag ggcttgagtg gatgggaaat attgatcctt actatggtgg tactagttac 180
aatcagaagt tcaagggcag agtcaccata accgcggaca cgtctacaga cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aacatgggac 300
tataggtacg acgacgggag ggcttactat gttatggact tctggggcca agggaccacg 360
gtcaccgtct cctcaggtgg aggcggttca ggcggaggtg gatccggcgg tggcggatcg 420
ggtggcggcg gatctgacat ccagatgacc cagtctccat cctccctgtc tgcatctgta 480
ggagacagag tcaccatcac ttgcaagtcc agccagagtg ttttatacag ctccaaccag 540
aagaactact tagcttggta tcagcagaaa ccagggaaag ttcctaagct cctgatctat 600
tgggcatcca ctagggaatc tggggtccca tctcggttca gtggcagtgg atctgggaca 660
gatttcactc tcaccatcag cagcctgcag cctgaagatg ttgcaactta ttactgtcat 720
caatacctct actcgtggac gtttggccag gggaccaagc tggagatcaa acggctcgag 780
cccaaatctt ctgacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgcgggt 840
gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080
aaggcgtacg cgtgcgcggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tccaagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagccgg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt taa 1473
<210> 231
<211> 490
<212> PRT
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0117 - 产生的人源化FUN1变体
<400> 231
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
100 105 110
Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
145 150 155 160
Gly Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Val Leu Tyr
165 170 175
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Lys Val Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
195 200 205
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
210 215 220
Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys His
225 230 235 240
Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
245 250 255
Lys Arg Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
260 265 270
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
340 345 350
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
355 360 365
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
485 490
<210> 232
<211> 1473
<212> DNA
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0118 – 产生的人源化FUN1变体
<400> 232
caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60
tcctgcaagg cttctggata caccttcacc gactacaaca tgaactgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaaat attgatcctt actatggtgg tactagttac 180
aatcagaagt tcaagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240
atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagatgggac 300
tataggtacg acgacgggag ggcttactat gttatggact tctggggcca agggaccacg 360
gtcaccgtct cctcaggtgg aggcggttca ggcggaggtg gatccggcgg tggcggatcg 420
ggtggcggcg gatctgacat ccagatgacc cagtctccat cctccctgtc tgcatctgta 480
ggagacagag tcaccatcac ttgcaagtcc agccagagtg ttttatacag ctccaaccag 540
aagaactact tagcttggta tcagcagaaa ccagggaaag ttcctaagct cctgatctat 600
tgggcatcca ctagggaatc tggggtccca tctcggttca gtggcagtgg atctgggaca 660
gatttcactc tcaccatcag cagcctgcag cctgaagatg ttgcaactta ttactgtcat 720
caatacctct actcgtggac gtttggccag gggaccaagc tggagatcaa acggctcgag 780
cccaaatctt ctgacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgcgggt 840
gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080
aaggcgtacg cgtgcgcggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tccaagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagccgg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt taa 1473
<210> 233
<211> 490
<212> PRT
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0118 – 产生的人源化FUN1变体
<400> 233
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
100 105 110
Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
145 150 155 160
Gly Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Val Leu Tyr
165 170 175
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Lys Val Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
195 200 205
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
210 215 220
Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys His
225 230 235 240
Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
245 250 255
Lys Arg Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
260 265 270
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
340 345 350
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
355 360 365
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
485 490
<210> 234
<211> 1473
<212> DNA
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0119 – 产生的人源化FUN1变体
<400> 234
caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt gactacaaca tgaactggat acgccaggct 120
ccagggaagg ggctggagtg ggtttcaaat attgatcctt actatggtgg tactagttac 180
aatcagaagt tcaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagatgggac 300
tataggtacg acgacgggag ggcttactat gttatggact tctggggcca agggaccacg 360
gtcaccgtct cctcaggtgg aggcggttca ggcggaggtg gatccggcgg tggcggatcg 420
ggtggcggcg gatctgacat ccagatgacc cagtctccat cctccctgtc tgcatctgta 480
ggagacagag tcaccatcac ttgcaagtcc agccagagtg ttttatacag ctccaaccag 540
aagaactact tagcttggta tcagcagaaa ccagggaaag ttcctaagct cctgatctat 600
tgggcatcca ctagggaatc tggggtccca tctcggttca gtggcagtgg atctgggaca 660
gatttcactc tcaccatcag cagcctgcag cctgaagatg ttgcaactta ttactgtcat 720
caatacctct actcgtggac gtttggccag gggaccaagc tggagatcaa acggctcgag 780
cccaaatctt ctgacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgcgggt 840
gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080
aaggcgtacg cgtgcgcggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tccaagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagccgg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt taa 1473
<210> 235
<211> 490
<212> PRT
<213> 人工序列
<220>
<223> 人源化FUN1 SMIP M0119 – 产生的人源化FUN1变体
<400> 235
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Asn Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
100 105 110
Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
145 150 155 160
Gly Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Val Leu Tyr
165 170 175
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Lys Val Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
195 200 205
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
210 215 220
Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys His
225 230 235 240
Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
245 250 255
Lys Arg Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
260 265 270
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
340 345 350
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
355 360 365
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
485 490
<210> 236
<211> 2013
<212> DNA
<213> 人工序列
<220>
<223> 编码人源化FUN1 M0115::IL10融合蛋白的核苷酸序列
<400> 236
caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60
tcctgcaagg cttctggata caccttcacc gactacaaca tgaactgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaaat attgatcctt actatggtgg tactagttac 180
aatcagaagt tcaagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240
atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagatgggac 300
tataggtacg acgacgggag ggcttactat gttatggact tctggggcca agggaccacg 360
gtcaccgtct cctcaggtgg aggcggttca ggcggaggtg gatccggcgg tggcggatcg 420
ggtggcggcg gatctgacat cgtgatgacc cagtctccag actccctggc tgtgtctctg 480
ggcgagaggg ccaccatcaa ctgcaagtcc agccagagtg ttttatacag ctccaaccag 540
aagaactact tagcttggta ccagcagaaa ccaggacagc ctcctaagct gctcatttac 600
tgggcatcta cccgggaatc cggggtccct gaccgattca gtggcagcgg gtctgggaca 660
gatttcactc tcaccatcag cagcctgcag gctgaagatg tggcagttta ttactgtcat 720
caatacctct actcgtggac gtttggccag gggaccaagc tggagatcaa acggctcgag 780
cccaaatctt ctgacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgcgggt 840
gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080
aaggcgtacg cgtgcgcggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tccaagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt aactcattat tcaaccaaga agttcaaatt 1500
cccttgaccg aaagttacag cccgaattct agcccaggcc agggcaccca gtctgagaac 1560
agctgcaccc acttcccagg caacctgcct aacatgcttc gagatctccg agatgccttc 1620
agcagagtga agactttctt tcaaatgaag gatcagctgg acaacttgtt gttaaaggag 1680
tccttgctgg aggactttaa gggttacctg ggttgccaag ccttgtctga gatgatccag 1740
ttttacctgg aggaggtgat gccccaagct gagaaccaag acccagacat caaggcgcat 1800
gtgaactccc tgggggagaa cctgaagacc ctcaggctga ggctacggcg ctgtcatcga 1860
tttcttccct gtgaaaacaa gagcaaggcc gtggagcagg tgaagaatgc ctttaataag 1920
ctccaagaga aaggcatcta caaagccatg agtgagtttg acatcttcat caactacata 1980
gaagcctaca tgacaatgaa gatacgaaac taa 2013
<210> 237
<211> 670
<212> PRT
<213> 人工序列
<220>
<223> 人源化FUN1 M0115::IL10融合蛋白
<400> 237
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
100 105 110
Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
145 150 155 160
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
165 170 175
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
195 200 205
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
210 215 220
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His
225 230 235 240
Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
245 250 255
Lys Arg Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
260 265 270
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
340 345 350
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
355 360 365
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln
485 490 495
Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Ser Pro
500 505 510
Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn
515 520 525
Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys
530 535 540
Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu
545 550 555 560
Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser
565 570 575
Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn
580 585 590
Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu
595 600 605
Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys
610 615 620
Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys
625 630 635 640
Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe
645 650 655
Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
660 665 670
<210> 238
<211> 1443
<212> DNA
<213> 人工序列
<220>
<223> 抗小鼠CD86 (GL1) SMIP
<400> 238
gaggtgcagc tggtggagtc tgctggaggt ttagtgcagc ctggaaggtc cctaaaactc 60
tcctgtgcag cctcaggatt cactttcagt gactattaca tggcctgggt ccgccaggct 120
ccaacgaagg ggctggagtg ggtcgcaacc attagtcatg acggtagtag tacttactat 180
cgagactccg tgaagggccg attcacaatc tccagagata atgcaaaaac catcctatac 240
ctgcaaatgg acagtctgag gtctgaggac acggccactt atttctgtgc aagacagaat 300
attatggatt attggggcca aggagtcatg gtcacagtct cctcaggggg tggaggctct 360
ggtggcggtg gctctggcgg aggtggatcc ggtggcggcg gatctgacat tgtgatgacc 420
cagactccat cctcccaggc tgtgtcagca ggggagaagg tcactatgag ctgcaagtcc 480
agtcagagtc ttttgtacag tagagaccag agtaactact tggcctggta ccagcagaaa 540
cctgggcagt ctcctaaact acttatctac ttggcatcca ctagggaatc aggggtccct 600
gatcgcttca taggcagtgg ctctgggaca gacttcactc tgaccatcag cagtgtgcag 660
gctgaagatc tggcagatta ttactgtcag cagcattaca actatccgct cacgttcggt 720
tctgggacca agctagaaat caaactcgag cccaaatctt ctgacaaaac tcacacatgc 780
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 840
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 900
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 960
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 1020
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 1080
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1140
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 1200
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 1260
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1320
tacagcaagc tcaccgtgga caagagccgg tggcagcagg ggaacgtctt ctcatgctcc 1380
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1440
taa 1443
<210> 239
<211> 480
<212> PRT
<213> 人工序列
<220>
<223> 抗小鼠CD86 (GL1) SMIP
<400> 239
Glu Val Gln Leu Val Glu Ser Ala Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser His Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Ile Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Gln Asn Ile Met Asp Tyr Trp Gly Gln Gly Val Met Val Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro Ser
130 135 140
Ser Gln Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser
145 150 155 160
Ser Gln Ser Leu Leu Tyr Ser Arg Asp Gln Ser Asn Tyr Leu Ala Trp
165 170 175
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Leu Ala
180 185 190
Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser
195 200 205
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu
210 215 220
Ala Asp Tyr Tyr Cys Gln Gln His Tyr Asn Tyr Pro Leu Thr Phe Gly
225 230 235 240
Ser Gly Thr Lys Leu Glu Ile Lys Leu Glu Pro Lys Ser Ser Asp Lys
245 250 255
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
260 265 270
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
275 280 285
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
290 295 300
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
305 310 315 320
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
325 330 335
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala
340 345 350
Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
355 360 365
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
370 375 380
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
385 390 395 400
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
405 410 415
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
420 425 430
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
435 440 445
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
450 455 460
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
465 470 475 480
<210> 240
<211> 345
<212> DNA
<213> 人工序列
<220>
<223> 编码抗小鼠CD86 (GL1)重链的核苷酸序列
<400> 240
gaggtgcagc tggtggagtc tgctggaggt ttagtgcagc ctggaaggtc cctaaaactc 60
tcctgtgcag cctcaggatt cactttcagt gactattaca tggcctgggt ccgccaggct 120
ccaacgaagg ggctggagtg ggtcgcaacc attagtcatg acggtagtag tacttactat 180
cgagactccg tgaagggccg attcacaatc tccagagata atgcaaaaac catcctatac 240
ctgcaaatgg acagtctgag gtctgaggac acggccactt atttctgtgc aagacagaat 300
attatggatt attggggcca aggagtcatg gtcacagtct cctca 345
<210> 241
<211> 115
<212> PRT
<213> 人工序列
<220>
<223> 抗CD86 (GL1)重链
<400> 241
Glu Val Gln Leu Val Glu Ser Ala Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser His Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Ile Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Gln Asn Ile Met Asp Tyr Trp Gly Gln Gly Val Met Val Thr
100 105 110
Val Ser Ser
115
<210> 242
<211> 339
<212> DNA
<213> 人工序列
<220>
<223> 编码抗小鼠CD86 (GL1)轻链的核苷酸序列
<400> 242
gacattgtga tgacccagac tccatcctcc caggctgtgt cagcagggga gaaggtcact 60
atgagctgca agtccagtca gagtcttttg tacagtagag accagagtaa ctacttggcc 120
tggtaccagc agaaacctgg gcagtctcct aaactactta tctacttggc atccactagg 180
gaatcagggg tccctgatcg cttcataggc agtggctctg ggacagactt cactctgacc 240
atcagcagtg tgcaggctga agatctggca gattattact gtcagcagca ttacaactat 300
ccgctcacgt tcggttctgg gaccaagcta gaaatcaaa 339
<210> 243
<211> 113
<212> PRT
<213> 人工序列
<220>
<223> 抗CD86 (GL1)轻链
<400> 243
Asp Ile Val Met Thr Gln Thr Pro Ser Ser Gln Ala Val Ser Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Arg Asp Gln Ser Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Leu Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Tyr Cys Gln Gln
85 90 95
His Tyr Asn Tyr Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 244
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 抗小鼠CD86 (GL1) scFv 接头
<400> 244
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 245
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 抗小鼠CD86 (GL1) HCDR1
<400> 245
Asp Tyr Tyr Met Ala
1 5
<210> 246
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 抗小鼠CD86 (GL1) HCDR2
<400> 246
Thr Ile Ser His Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 247
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 抗小鼠CD86 (GL1) HCDR3
<400> 247
Gln Asn Ile Met Asp Tyr
1 5
<210> 248
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 抗小鼠CD86 (GL1) LCDR1
<400> 248
Lys Ser Ser Gln Ser Leu Leu Tyr Ser Arg Asp Gln Ser Asn Tyr Leu
1 5 10 15
Ala
<210> 249
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 抗小鼠CD86 (GL1) LCDR2
<400> 249
Leu Ala Ser Thr Arg Glu Ser
1 5
<210> 250
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 抗小鼠CD86 (GL1) LCDR3
<400> 250
Gln Gln His Tyr Asn Tyr Pro Leu Thr
1 5
<210> 251
<211> 1983
<212> DNA
<213> 人工序列
<220>
<223> 编码小鼠抗CD86::IL10融合蛋白的核苷酸序列
<400> 251
gaggtgcagc tggtggagtc tgctggaggt ttagtgcagc ctggaaggtc cctaaaactc 60
tcctgtgcag cctcaggatt cactttcagt gactattaca tggcctgggt ccgccaggct 120
ccaacgaagg ggctggagtg ggtcgcaacc attagtcatg acggtagtag tacttactat 180
cgagactccg tgaagggccg attcacaatc tccagagata atgcaaaaac catcctatac 240
ctgcaaatgg acagtctgag gtctgaggac acggccactt atttctgtgc aagacagaat 300
attatggatt attggggcca aggagtcatg gtcacagtct cctcaggggg tggaggctct 360
ggtggcggtg gctctggcgg aggtggatcc ggtggcggcg gatctgacat tgtgatgacc 420
cagactccat cctcccaggc tgtgtcagca ggggagaagg tcactatgag ctgcaagtcc 480
agtcagagtc ttttgtacag tagagaccag agtaactact tggcctggta ccagcagaaa 540
cctgggcagt ctcctaaact acttatctac ttggcatcca ctagggaatc aggggtccct 600
gatcgcttca taggcagtgg ctctgggaca gacttcactc tgaccatcag cagtgtgcag 660
gctgaagatc tggcagatta ttactgtcag cagcattaca actatccgct cacgttcggt 720
tctgggacca agctagaaat caaactcgag cccaaatctt ctgacaaaac tcacacatgc 780
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 840
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 900
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 960
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 1020
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 1080
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1140
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 1200
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 1260
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1320
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1380
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1440
aactcattat tcaaccaaga agttcaaatt cccttgaccg aaagttacag cccgaattct 1500
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 1560
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 1620
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 1680
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 1740
gagaaccaag acccagacat caaggcgcat gtgaactccc tgggggagaa cctgaagacc 1800
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 1860
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 1920
agtgagtttg acatcttcat caactacata gaagcctaca tgacaatgaa gatacgaaac 1980
taa 1983
<210> 252
<211> 660
<212> PRT
<213> 人工序列
<220>
<223> 小鼠抗CD86::IL10融合蛋白
<400> 252
Glu Val Gln Leu Val Glu Ser Ala Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser His Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Ile Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Gln Asn Ile Met Asp Tyr Trp Gly Gln Gly Val Met Val Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro Ser
130 135 140
Ser Gln Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser
145 150 155 160
Ser Gln Ser Leu Leu Tyr Ser Arg Asp Gln Ser Asn Tyr Leu Ala Trp
165 170 175
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Leu Ala
180 185 190
Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser
195 200 205
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu
210 215 220
Ala Asp Tyr Tyr Cys Gln Gln His Tyr Asn Tyr Pro Leu Thr Phe Gly
225 230 235 240
Ser Gly Thr Lys Leu Glu Ile Lys Leu Glu Pro Lys Ser Ser Asp Lys
245 250 255
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
260 265 270
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
275 280 285
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
290 295 300
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
305 310 315 320
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
325 330 335
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala
340 345 350
Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
355 360 365
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
370 375 380
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
385 390 395 400
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
405 410 415
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
420 425 430
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
435 440 445
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
450 455 460
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
465 470 475 480
Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr
485 490 495
Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys
500 505 510
Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp
515 520 525
Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp
530 535 540
Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu
545 550 555 560
Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val
565 570 575
Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn
580 585 590
Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys
595 600 605
His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val
610 615 620
Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met
625 630 635 640
Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met
645 650 655
Lys Ile Arg Asn
660
<210> 253
<211> 2013
<212> DNA
<213> 人工序列
<220>
<223> 编码IL10::人源化Fun1 M0115融合蛋白的核苷酸序列
<400> 253
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacat caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggtaact cattattcaa ccaagaagtt 1200
caaattccct tgaccgaaag ttacagcccg aattctcagg tgcagctggt gcagtctggg 1260
gctgaggtga agaagcctgg ggcctcagtg aaggtctcct gcaaggcttc tggatacacc 1320
ttcaccgact acaacatgaa ctgggtgcga caggcccctg gacaagggct tgagtggatg 1380
ggaaatattg atccttacta tggtggtact agttacaatc agaagttcaa gggcagggtc 1440
accatgacca gggacacgtc catcagcaca gcctacatgg agctgagcag gctgagatct 1500
gacgacacgg ccgtgtatta ctgtgcgaga tgggactata ggtacgacga cgggagggct 1560
tactatgtta tggacttctg gggccaaggg accacggtca ccgtctcctc aggtggaggc 1620
ggttcaggcg gaggtggatc cggcggtggc ggatcgggtg gcggcggatc tgacatcgtg 1680
atgacccagt ctccagactc cctggctgtg tctctgggcg agagggccac catcaactgc 1740
aagtccagcc agagtgtttt atacagctcc aaccagaaga actacttagc ttggtaccag 1800
cagaaaccag gacagcctcc taagctgctc atttactggg catctacccg ggaatccggg 1860
gtccctgacc gattcagtgg cagcgggtct gggacagatt tcactctcac catcagcagc 1920
ctgcaggctg aagatgtggc agtttattac tgtcatcaat acctctactc gtggacgttt 1980
ggccagggga ccaagctgga gatcaaacgg taa 2013
<210> 254
<211> 670
<212> PRT
<213> 人工序列
<220>
<223> IL10::人源化Fun1 M0115融合蛋白
<400> 254
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val
385 390 395 400
Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Gln Val Gln Leu
405 410 415
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val
420 425 430
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Asn Met Asn Trp
435 440 445
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Asn Ile Asp
450 455 460
Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg Val
465 470 475 480
Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser
485 490 495
Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Asp
500 505 510
Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met Asp Phe Trp Gly
515 520 525
Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
530 535 540
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val
545 550 555 560
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala
565 570 575
Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Gln
580 585 590
Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys
595 600 605
Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg
610 615 620
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
625 630 635 640
Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln Tyr Leu Tyr
645 650 655
Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
660 665 670
<210> 255
<211> 1983
<212> DNA
<213> 人工序列
<220>
<223> 编码IL10::抗小鼠CD86融合蛋白的核苷酸序列
<400> 255
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacat caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggtaact cattattcaa ccaagaagtt 1200
caaattccct tgaccgaaag ttacagcccg aattctgagg tgcagctggt ggagtctgct 1260
ggaggtttag tgcagcctgg aaggtcccta aaactctcct gtgcagcctc aggattcact 1320
ttcagtgact attacatggc ctgggtccgc caggctccaa cgaaggggct ggagtgggtc 1380
gcaaccatta gtcatgacgg tagtagtact tactatcgag actccgtgaa gggccgattc 1440
acaatctcca gagataatgc aaaaaccatc ctatacctgc aaatggacag tctgaggtct 1500
gaggacacgg ccacttattt ctgtgcaaga cagaatatta tggattattg gggccaagga 1560
gtcatggtca cagtctcctc agggggtgga ggctctggtg gcggtggctc tggcggaggt 1620
ggatccggtg gcggcggatc tgacattgtg atgacccaga ctccatcctc ccaggctgtg 1680
tcagcagggg agaaggtcac tatgagctgc aagtccagtc agagtctttt gtacagtaga 1740
gaccagagta actacttggc ctggtaccag cagaaacctg ggcagtctcc taaactactt 1800
atctacttgg catccactag ggaatcaggg gtccctgatc gcttcatagg cagtggctct 1860
gggacagact tcactctgac catcagcagt gtgcaggctg aagatctggc agattattac 1920
tgtcagcagc attacaacta tccgctcacg ttcggttctg ggaccaagct agaaatcaaa 1980
taa 1983
<210> 256
<211> 660
<212> PRT
<213> 人工序列
<220>
<223> IL10::抗小鼠CD86融合蛋白
<400> 256
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val
385 390 395 400
Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Glu Val Gln Leu
405 410 415
Val Glu Ser Ala Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Lys Leu
420 425 430
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Ala Trp
435 440 445
Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val Ala Thr Ile Ser
450 455 460
His Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe
465 470 475 480
Thr Ile Ser Arg Asp Asn Ala Lys Thr Ile Leu Tyr Leu Gln Met Asp
485 490 495
Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gln Asn
500 505 510
Ile Met Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser Gly
515 520 525
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
530 535 540
Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro Ser Ser Gln Ala Val
545 550 555 560
Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu
565 570 575
Leu Tyr Ser Arg Asp Gln Ser Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
580 585 590
Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Leu Ala Ser Thr Arg Glu
595 600 605
Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe
610 615 620
Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Tyr
625 630 635 640
Cys Gln Gln His Tyr Asn Tyr Pro Leu Thr Phe Gly Ser Gly Thr Lys
645 650 655
Leu Glu Ile Lys
660
<210> 257
<211> 2016
<212> DNA
<213> 人工序列
<220>
<223> 编码含H75接头的IL10 I87A::人源化Fun1 M0115融合蛋白的核苷酸序列
<400> 257
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacgc caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggtcaga ggcacaacaa ttcttccctg 1200
aatacaggaa ctcagatggc aggtcattct ccgaattctc aggtgcagct ggtgcagtct 1260
ggggctgagg tgaagaagcc tggggcctca gtgaaggtct cctgcaaggc ttctggatac 1320
accttcaccg actacaacat gaactgggtg cgacaggccc ctggacaagg gcttgagtgg 1380
atgggaaata ttgatcctta ctatggtggt actagttaca atcagaagtt caagggcagg 1440
gtcaccatga ccagggacac gtccatcagc acagcctaca tggagctgag caggctgaga 1500
tctgacgaca cggccgtgta ttactgtgcg agatgggact ataggtacga cgacgggagg 1560
gcttactatg ttatggactt ctggggccaa gggaccacgg tcaccgtctc ctcaggtgga 1620
ggcggttcag gcggaggtgg atccggcggt ggcggatcgg gtggcggcgg atctgacatc 1680
gtgatgaccc agtctccaga ctccctggct gtgtctctgg gcgagagggc caccatcaac 1740
tgcaagtcca gccagagtgt tttatacagc tccaaccaga agaactactt agcttggtac 1800
cagcagaaac caggacagcc tcctaagctg ctcatttact gggcatctac ccgggaatcc 1860
ggggtccctg accgattcag tggcagcggg tctgggacag atttcactct caccatcagc 1920
agcctgcagg ctgaagatgt ggcagtttat tactgtcatc aatacctcta ctcgtggacg 1980
tttggccagg ggaccaagct ggagatcaaa cggtaa 2016
<210> 258
<211> 671
<212> PRT
<213> 人工序列
<220>
<223> 含H75接头的IL10 I87A::人源化Fun1 M0115融合蛋白
<400> 258
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu
385 390 395 400
Asn Thr Gly Thr Gln Met Ala Gly His Ser Pro Asn Ser Gln Val Gln
405 410 415
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
420 425 430
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Asn Met Asn
435 440 445
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Asn Ile
450 455 460
Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg
465 470 475 480
Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu
485 490 495
Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp
500 505 510
Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met Asp Phe Trp
515 520 525
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile
545 550 555 560
Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg
565 570 575
Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn
580 585 590
Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
595 600 605
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp
610 615 620
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
625 630 635 640
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln Tyr Leu
645 650 655
Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
660 665 670
<210> 259
<211> 1986
<212> DNA
<213> 人工序列
<220>
<223> 编码含H75接头的IL10 I87A::抗小鼠CD86融合蛋白的核苷酸序列
<400> 259
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacgc caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggtcaga ggcacaacaa ttcttccctg 1200
aatacaggaa ctcagatggc aggtcattct ccgaattctg aggtgcagct ggtggagtct 1260
gctggaggtt tagtgcagcc tggaaggtcc ctaaaactct cctgtgcagc ctcaggattc 1320
actttcagtg actattacat ggcctgggtc cgccaggctc caacgaaggg gctggagtgg 1380
gtcgcaacca ttagtcatga cggtagtagt acttactatc gagactccgt gaagggccga 1440
ttcacaatct ccagagataa tgcaaaaacc atcctatacc tgcaaatgga cagtctgagg 1500
tctgaggaca cggccactta tttctgtgca agacagaata ttatggatta ttggggccaa 1560
ggagtcatgg tcacagtctc ctcagggggt ggaggctctg gtggcggtgg ctctggcgga 1620
ggtggatccg gtggcggcgg atctgacatt gtgatgaccc agactccatc ctcccaggct 1680
gtgtcagcag gggagaaggt cactatgagc tgcaagtcca gtcagagtct tttgtacagt 1740
agagaccaga gtaactactt ggcctggtac cagcagaaac ctgggcagtc tcctaaacta 1800
cttatctact tggcatccac tagggaatca ggggtccctg atcgcttcat aggcagtggc 1860
tctgggacag acttcactct gaccatcagc agtgtgcagg ctgaagatct ggcagattat 1920
tactgtcagc agcattacaa ctatccgctc acgttcggtt ctgggaccaa gctagaaatc 1980
aaataa 1986
<210> 260
<211> 661
<212> PRT
<213> 人工序列
<220>
<223> 含H75接头的IL10 I87A::抗小鼠CD86融合蛋白
<400> 260
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu
385 390 395 400
Asn Thr Gly Thr Gln Met Ala Gly His Ser Pro Asn Ser Glu Val Gln
405 410 415
Leu Val Glu Ser Ala Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Lys
420 425 430
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Ala
435 440 445
Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val Ala Thr Ile
450 455 460
Ser His Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg
465 470 475 480
Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Ile Leu Tyr Leu Gln Met
485 490 495
Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gln
500 505 510
Asn Ile Met Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro Ser Ser Gln Ala
545 550 555 560
Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser
565 570 575
Leu Leu Tyr Ser Arg Asp Gln Ser Asn Tyr Leu Ala Trp Tyr Gln Gln
580 585 590
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Leu Ala Ser Thr Arg
595 600 605
Glu Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp
610 615 620
Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr
625 630 635 640
Tyr Cys Gln Gln His Tyr Asn Tyr Pro Leu Thr Phe Gly Ser Gly Thr
645 650 655
Lys Leu Glu Ile Lys
660
<210> 261
<211> 1614
<212> DNA
<213> 人工序列
<220>
<223> 编码含H75接头的CTLA4::IL10 I87A融合蛋白的核苷酸序列
<400> 261
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtca gaggcacaac 1080
aattcttccc tgaatacagg aactcagatg gcaggtcatt ctccgaattc tagcccaggc 1140
cagggcaccc agtctgagaa cagctgcacc cacttcccag gcaacctgcc taacatgctt 1200
cgagatctcc gagatgcctt cagcagagtg aagactttct ttcaaatgaa ggatcagctg 1260
gacaacttgt tgttaaagga gtccttgctg gaggacttta agggttacct gggttgccaa 1320
gccttgtctg agatgatcca gttttacctg gaggaggtga tgccccaagc tgagaaccaa 1380
gacccagacg ccaaggcgca tgtgaactcc ctgggggaga acctgaagac cctcaggctg 1440
aggctacggc gctgtcatcg atttcttccc tgtgaaaaca agagcaaggc cgtggagcag 1500
gtgaagaatg cctttaataa gctccaagag aaaggcatct acaaagccat gagtgagttt 1560
gacatcttca tcaactacat agaagcctac atgacaatga agatacgaaa ctaa 1614
<210> 262
<211> 537
<212> PRT
<213> 人工序列
<220>
<223> 含H75接头的CTLA4::IL10 I87A融合蛋白
<400> 262
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Gly Thr
355 360 365
Gln Met Ala Gly His Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln
370 375 380
Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu
385 390 395 400
Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met
405 410 415
Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp
420 425 430
Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe
435 440 445
Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ala
450 455 460
Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu
465 470 475 480
Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys
485 490 495
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly
500 505 510
Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu
515 520 525
Ala Tyr Met Thr Met Lys Ile Arg Asn
530 535
<210> 263
<211> 1380
<212> DNA
<213> 人工序列
<220>
<223> 淋巴毒素β受体(LTβR) - Ig合成分子
<400> 263
atgctcctgc cttgggccac ctctgccccc ggcctggcct gggggcctct ggtgctgggc 60
ctcttcgggc tcctggcagc atcgcagccc caggcggtgc ctccatatgc gtcggagaac 120
cagacctgca gggaccagga aaaggaatac tatgagcccc agcaccgcat ctgctgctcc 180
cgctgcccgc caggcaccta tgtctcagct aaatgtagcc gcatccggga cacagtttgt 240
gccacatgtg ccgagaactc ctacaacgag cactggaact acctgaccat ctgccagctg 300
tgccgcccct gtgacccagt gatgggccta gaggagattg ccccctgcac aagcaaacgg 360
aagacccagt gccgctgcca gccgggaatg ttctgtgctg cctgggccct agagtgcaca 420
cactgcgagc tactttctga ctgcccgcct ggcactgaag ccgagctcaa agatgaagtt 480
gggaagggta acaaccactg cgtcccctgc aaggcagggc acttccagaa tacctcctcc 540
cccagcgccc gctgccagcc ccacaccagg tgtgagaacc aaggtctggt ggaggcagct 600
ccaggcactg cccagtccga cacaacctgc aaaaatccat tagagccact gcccccagag 660
atgtcaggaa ccatgctgat gctcgagccc aaatcttctg acaaaactca cacatgccca 720
ccgtgcccag cacctgaagc cgcgggtgca ccgtcagtct tcctcttccc cccaaaaccc 780
aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 840
cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 900
aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 960
gtcctgcacc aggactggct gaatggcaag gcgtacgcgt gcgcggtctc caacaaagcc 1020
ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1080
gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1140
ctggtcaaag gcttctatcc aagcgacatc gccgtggagt gggagagcaa tgggcagccg 1200
gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1260
agcaagctca ccgtggacaa gagccggtgg cagcagggga acgtcttctc atgctccgtg 1320
atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggttaa 1380
<210> 264
<211> 459
<212> PRT
<213> 人工序列
<220>
<223> 淋巴毒素β受体(LTβR) – Ig合成分子
<400> 264
Met Leu Leu Pro Trp Ala Thr Ser Ala Pro Gly Leu Ala Trp Gly Pro
1 5 10 15
Leu Val Leu Gly Leu Phe Gly Leu Leu Ala Ala Ser Gln Pro Gln Ala
20 25 30
Val Pro Pro Tyr Ala Ser Glu Asn Gln Thr Cys Arg Asp Gln Glu Lys
35 40 45
Glu Tyr Tyr Glu Pro Gln His Arg Ile Cys Cys Ser Arg Cys Pro Pro
50 55 60
Gly Thr Tyr Val Ser Ala Lys Cys Ser Arg Ile Arg Asp Thr Val Cys
65 70 75 80
Ala Thr Cys Ala Glu Asn Ser Tyr Asn Glu His Trp Asn Tyr Leu Thr
85 90 95
Ile Cys Gln Leu Cys Arg Pro Cys Asp Pro Val Met Gly Leu Glu Glu
100 105 110
Ile Ala Pro Cys Thr Ser Lys Arg Lys Thr Gln Cys Arg Cys Gln Pro
115 120 125
Gly Met Phe Cys Ala Ala Trp Ala Leu Glu Cys Thr His Cys Glu Leu
130 135 140
Leu Ser Asp Cys Pro Pro Gly Thr Glu Ala Glu Leu Lys Asp Glu Val
145 150 155 160
Gly Lys Gly Asn Asn His Cys Val Pro Cys Lys Ala Gly His Phe Gln
165 170 175
Asn Thr Ser Ser Pro Ser Ala Arg Cys Gln Pro His Thr Arg Cys Glu
180 185 190
Asn Gln Gly Leu Val Glu Ala Ala Pro Gly Thr Ala Gln Ser Asp Thr
195 200 205
Thr Cys Lys Asn Pro Leu Glu Pro Leu Pro Pro Glu Met Ser Gly Thr
210 215 220
Met Leu Met Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
225 230 235 240
Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe
245 250 255
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
260 265 270
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
275 280 285
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
290 295 300
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
305 310 315 320
Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val
325 330 335
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
340 345 350
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
355 360 365
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
370 375 380
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
385 390 395 400
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
405 410 415
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
420 425 430
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
435 440 445
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
450 455
<210> 265
<211> 2217
<212> DNA
<213> 人工序列
<220>
<223> 编码LTβR::M0115融合蛋白的核苷酸序列
<400> 265
atgctcctgc cttgggccac ctctgccccc ggcctggcct gggggcctct ggtgctgggc 60
ctcttcgggc tcctggcagc atcgcagccc caggcggtgc ctccatatgc gtcggagaac 120
cagacctgca gggaccagga aaaggaatac tatgagcccc agcaccgcat ctgctgctcc 180
cgctgcccgc caggcaccta tgtctcagct aaatgtagcc gcatccggga cacagtttgt 240
gccacatgtg ccgagaactc ctacaacgag cactggaact acctgaccat ctgccagctg 300
tgccgcccct gtgacccagt gatgggccta gaggagattg ccccctgcac aagcaaacgg 360
aagacccagt gccgctgcca gccgggaatg ttctgtgctg cctgggccct agagtgcaca 420
cactgcgagc tactttctga ctgcccgcct ggcactgaag ccgagctcaa agatgaagtt 480
gggaagggta acaaccactg cgtcccctgc aaggcagggc acttccagaa tacctcctcc 540
cccagcgccc gctgccagcc ccacaccagg tgtgagaacc aaggtctggt ggaggcagct 600
ccaggcactg cccagtccga cacaacctgc aaaaatccat tagagccact gcccccagag 660
atgtcaggaa ccatgctgat gctcgagccc aaatcttctg acaaaactca cacatgccca 720
ccgtgcccag cacctgaagc cgcgggtgca ccgtcagtct tcctcttccc cccaaaaccc 780
aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 840
cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 900
aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 960
gtcctgcacc aggactggct gaatggcaag gcgtacgcgt gcgcggtctc caacaaagcc 1020
ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1080
gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1140
ctggtcaaag gcttctatcc aagcgacatc gccgtggagt gggagagcaa tgggcagccg 1200
gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1260
agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1320
atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtcag 1380
aggcacaaca attcttccct gaatacagga actcagatgg caggtcattc tccgaattct 1440
caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 1500
tcctgcaagg cttctggata caccttcacc gactacaaca tgaactgggt gcgacaggcc 1560
cctggacaag ggcttgagtg gatgggaaat attgatcctt actatggtgg tactagttac 1620
aatcagaagt tcaagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 1680
atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagatgggac 1740
tataggtacg acgacgggag ggcttactat gttatggact tctggggcca agggaccacg 1800
gtcaccgtct cctcaggtgg aggcggttca ggcggaggtg gatccggcgg tggcggatcg 1860
ggtggcggcg gatctgacat cgtgatgacc cagtctccag actccctggc tgtgtctctg 1920
ggcgagaggg ccaccatcaa ctgcaagtcc agccagagtg ttttatacag ctccaaccag 1980
aagaactact tagcttggta ccagcagaaa ccaggacagc ctcctaagct gctcatttac 2040
tgggcatcta cccgggaatc cggggtccct gaccgattca gtggcagcgg gtctgggaca 2100
gatttcactc tcaccatcag cagcctgcag gctgaagatg tggcagttta ttactgtcat 2160
caatacctct actcgtggac gtttggccag gggaccaagc tggagatcaa acggtaa 2217
<210> 266
<211> 738
<212> PRT
<213> 人工序列
<220>
<223> LTβR::M0115融合蛋白
<400> 266
Met Leu Leu Pro Trp Ala Thr Ser Ala Pro Gly Leu Ala Trp Gly Pro
1 5 10 15
Leu Val Leu Gly Leu Phe Gly Leu Leu Ala Ala Ser Gln Pro Gln Ala
20 25 30
Val Pro Pro Tyr Ala Ser Glu Asn Gln Thr Cys Arg Asp Gln Glu Lys
35 40 45
Glu Tyr Tyr Glu Pro Gln His Arg Ile Cys Cys Ser Arg Cys Pro Pro
50 55 60
Gly Thr Tyr Val Ser Ala Lys Cys Ser Arg Ile Arg Asp Thr Val Cys
65 70 75 80
Ala Thr Cys Ala Glu Asn Ser Tyr Asn Glu His Trp Asn Tyr Leu Thr
85 90 95
Ile Cys Gln Leu Cys Arg Pro Cys Asp Pro Val Met Gly Leu Glu Glu
100 105 110
Ile Ala Pro Cys Thr Ser Lys Arg Lys Thr Gln Cys Arg Cys Gln Pro
115 120 125
Gly Met Phe Cys Ala Ala Trp Ala Leu Glu Cys Thr His Cys Glu Leu
130 135 140
Leu Ser Asp Cys Pro Pro Gly Thr Glu Ala Glu Leu Lys Asp Glu Val
145 150 155 160
Gly Lys Gly Asn Asn His Cys Val Pro Cys Lys Ala Gly His Phe Gln
165 170 175
Asn Thr Ser Ser Pro Ser Ala Arg Cys Gln Pro His Thr Arg Cys Glu
180 185 190
Asn Gln Gly Leu Val Glu Ala Ala Pro Gly Thr Ala Gln Ser Asp Thr
195 200 205
Thr Cys Lys Asn Pro Leu Glu Pro Leu Pro Pro Glu Met Ser Gly Thr
210 215 220
Met Leu Met Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
225 230 235 240
Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe
245 250 255
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
260 265 270
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
275 280 285
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
290 295 300
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
305 310 315 320
Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val
325 330 335
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
340 345 350
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
355 360 365
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
370 375 380
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
385 390 395 400
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
405 410 415
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
420 425 430
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
435 440 445
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gln Arg His Asn Asn
450 455 460
Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Gly His Ser Pro Asn Ser
465 470 475 480
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
485 490 495
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
500 505 510
Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
515 520 525
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
530 535 540
Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
545 550 555 560
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
565 570 575
Ala Arg Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
580 585 590
Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
595 600 605
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
610 615 620
Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
625 630 635 640
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
645 650 655
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
660 665 670
Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
675 680 685
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
690 695 700
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His
705 710 715 720
Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
725 730 735
Lys Arg
<210> 267
<211> 1359
<212> DNA
<213> 人工序列
<220>
<223> PDL1 Ig合成分子
<400> 267
tttactgtca cggttcccaa ggacctatat gtggtagagt atggtagcaa tatgacaatt 60
gaatgcaaat tcccagtaga aaaacaatta gacctggctg cactaattgt ctattgggaa 120
atggaggata agaacattat tcaatttgtg catggagagg aagacctgaa ggttcagcat 180
agtagctaca gacagagggc ccggctgttg aaggaccagc tctccctggg aaatgctgca 240
cttcagatca cagatgtgaa attgcaggat gcaggggtgt accgctgcat gatcagctat 300
ggtggtgccg actacaagcg aattactgtg aaagtcaatg ccccatacaa caaaatcaac 360
caaagaattt tggttgtgga tccagtcacc tctgaacatg aactgacatg tcaggctgag 420
ggctacccca aggccgaagt catctggaca agcagtgacc atcaagtcct gagtggtaag 480
accaccacca ccaattccaa gagagaggag aagcttttca atgtgaccag cacactgaga 540
atcaacacaa caactaatga gattttctac tgcactttta ggagattaga tcctgaggaa 600
aaccatacag ctgaattggt catcccagaa ctacctctgg cacatcctcc aaatgaaagg 660
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 720
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agccggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggttaa 1359
<210> 268
<211> 452
<212> PRT
<213> 人工序列
<220>
<223> PDL1 Ig合成分子
<400> 268
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Leu Glu Pro Lys
210 215 220
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
225 230 235 240
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
305 310 315 320
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
355 360 365
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
435 440 445
Leu Ser Pro Gly
450
<210> 269
<211> 1302
<212> DNA
<213> 人工序列
<220>
<223> PDL2 Ig合成分子
<400> 269
ttattcacag tgacagtccc taaggaactg tacataatag agcatggcag caatgtgacc 60
ctggaatgca actttgacac tggaagtcat gtgaaccttg gagcaataac agccagtttg 120
caaaaggtgg aaaatgatac atccccacac cgtgaaagag ccactttgct ggaggagcag 180
ctgcccttag ggaaggcctc gttccacata cctcaagtcc aagtgaggga cgaaggacag 240
taccaatgca taatcatcta tggggtcgcc tgggactaca agtacctgac tctgaaagtc 300
aaagcttcct acaggaaaat aaacactcac atcctaaagg ttccagaaac agatgaggta 360
gagctcacct gccaggctac aggttatcct ctggcagaag tatcctggcc aaacgtcagc 420
gttcctgcca acaccagcca ctccaggacc cctgaaggcc tctaccaggt caccagtgtt 480
ctgcgcctaa agccaccccc tggcagaaac ttcagctgtg tgttctggaa tactcacgtg 540
agggaactta ctttggccag cattgacctt caaagtcaga tggaacccag gacccatcca 600
actctcgagc ccaaatcttc tgacaaaact cacacatgcc caccgtgccc agcacctgaa 660
gccgcgggtg caccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 720
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 780
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 840
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 900
ctgaatggca aggcgtacgc gtgcgcggtc tccaacaaag ccctcccagc ccccatcgag 960
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1020
tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1080
ccaagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1140
acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1200
aagagccggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1260
aaccactaca cgcagaagag cctctccctg tctccgggtt aa 1302
<210> 270
<211> 433
<212> PRT
<213> 人工序列
<220>
<223> PDL2 Ig合成分子
<400> 270
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Leu Glu Pro Lys Ser Ser Asp
195 200 205
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala
210 215 220
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
225 230 235 240
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
245 250 255
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
260 265 270
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
275 280 285
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
290 295 300
Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
305 310 315 320
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
325 330 335
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
340 345 350
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
355 360 365
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
370 375 380
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
385 390 395 400
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
405 410 415
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
420 425 430
Gly
<210> 271
<211> 534
<212> DNA
<213> 人工序列
<220>
<223> (GITR-L)-AFH合成分子
<400> 271
tctggtagcc atcatcatca tcatcacctg aacgacatct tcgaggctca gaaaatcgaa 60
tggcacgaag attacaagga tgacgacgat aaggattaca aggatgacga cgataaggat 120
tacaaggatg acgacgataa gctcgagcaa ttagagactg ctaaggagcc ctgtatggct 180
aagtttggac cattaccctc aaaatggcaa atggcatctt ctgaacctcc ttgcgtgaat 240
aaggtgtctg actggaagct ggagatactt cagaatggct tatatttaat ttatggccaa 300
gtggctccca atgcaaacta caatgatgta gctccttttg aggtgcggct gtataaaaac 360
aaagacatga tacaaactct aacaaacaaa tctaaaatcc aaaatgtagg agggacttat 420
gaattgcatg ttggggacac catagacttg atattcaact ctgagcatca ggttctaaaa 480
aataatacat actggggtat cattttacta gcaaatcccc aattcatctc ctaa 534
<210> 272
<211> 177
<212> PRT
<213> 人工序列
<220>
<223> (GITR-L)-AFH合成分子
<400> 272
Ser Gly Ser His His His His His His Leu Asn Asp Ile Phe Glu Ala
1 5 10 15
Gln Lys Ile Glu Trp His Glu Asp Tyr Lys Asp Asp Asp Asp Lys Asp
20 25 30
Tyr Lys Asp Asp Asp Asp Lys Asp Tyr Lys Asp Asp Asp Asp Lys Leu
35 40 45
Glu Gln Leu Glu Thr Ala Lys Glu Pro Cys Met Ala Lys Phe Gly Pro
50 55 60
Leu Pro Ser Lys Trp Gln Met Ala Ser Ser Glu Pro Pro Cys Val Asn
65 70 75 80
Lys Val Ser Asp Trp Lys Leu Glu Ile Leu Gln Asn Gly Leu Tyr Leu
85 90 95
Ile Tyr Gly Gln Val Ala Pro Asn Ala Asn Tyr Asn Asp Val Ala Pro
100 105 110
Phe Glu Val Arg Leu Tyr Lys Asn Lys Asp Met Ile Gln Thr Leu Thr
115 120 125
Asn Lys Ser Lys Ile Gln Asn Val Gly Gly Thr Tyr Glu Leu His Val
130 135 140
Gly Asp Thr Ile Asp Leu Ile Phe Asn Ser Glu His Gln Val Leu Lys
145 150 155 160
Asn Asn Thr Tyr Trp Gly Ile Ile Leu Leu Ala Asn Pro Gln Phe Ile
165 170 175
Ser
<210> 273
<211> 1281
<212> DNA
<213> 人工序列
<220>
<223> mIg LIGHT合成分子
<400> 273
agctccccca gagtgcccat aacacagaac ccctgtcctc cactcaaaga gtgtccccca 60
tgcgcagctc cagacgcagc gggtgcgcca tccgtcttca tcttccctcc aaagatcaag 120
gatgtactca tgatctccct gagccccatg gtcacatgtg tggtggtgga tgtgagcgag 180
gatgacccag acgtccagat cagctggttt gtgaacaacg tggaagtaca cacagctcag 240
acacaaaccc atagagagga ttacaacagt actctccggg tggtcagtgc cctccccatc 300
cagcaccagg actggatgag tggcaaggcg ttcgcatgcg cggtcaacaa cagagccctc 360
ccatccccca tcgagaaaac catctcaaaa cccagagggc cagtaagagc tccacaggta 420
tatgtcttgc ctccaccagc agaagagatg actaagaaag agttcagtct gacctgcatg 480
atcacaggct tcttacctgc cgaaattgct gtggactgga ccagcaatgg gcgtacagag 540
caaaactaca agaacaccgc aacagtcctg gactctgatg gttcttactt catgtacagc 600
aagctcagag tacaaaagag cacttgggaa agaggaagtc ttttcgcctg ctcagtggtc 660
cacgagggtc tgcacaatca ccttacgact aagaccatct cccggtctct gggtaaaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttacagccc gaattctgac 780
ggacctgcag gctcctggga gcagctgata caagagcgaa ggtctcacga ggtcaaccca 840
gcagcgcatc tcacaggggc caactccagc ttgaccggca gcggggggcc gctgttatgg 900
gagactcagc tgggcctggc cttcctgagg ggcctcagct accacgatgg ggcccttgtg 960
gtcaccaaag ctggctacta ctacatctac tccaaggtgc agctgggcgg tgtgggctgc 1020
ccgctgggcc tggccagcac catcacccac ggcctctaca agcgcacacc ccgctacccc 1080
gaggagctgg agctgttggt cagccagcag tcaccctgcg gacgggccac cagcagctcc 1140
cgggtctggt gggacagcag cttcctgggt ggtgtggtac acctggaggc tggggagaag 1200
gtggtcgtcc gtgtgctgga tgaacgcctg gttcgactgc gtgatggtac ccggtcttac 1260
ttcggggctt tcatggtgta g 1281
<210> 274
<211> 426
<212> PRT
<213> 人工序列
<220>
<223> mIg LIGHT合成分子
<400> 274
Ser Ser Pro Arg Val Pro Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys
1 5 10 15
Glu Cys Pro Pro Cys Ala Ala Pro Asp Ala Ala Gly Ala Pro Ser Val
20 25 30
Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser
35 40 45
Pro Met Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp
50 55 60
Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln
65 70 75 80
Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser
85 90 95
Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Ala Phe Ala
100 105 110
Cys Ala Val Asn Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile
115 120 125
Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro
130 135 140
Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys Met
145 150 155 160
Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn
165 170 175
Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser
180 185 190
Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr
195 200 205
Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val Val His Glu Gly Leu
210 215 220
His Asn His Leu Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly Lys Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Ser
245 250 255
Pro Asn Ser Asp Gly Pro Ala Gly Ser Trp Glu Gln Leu Ile Gln Glu
260 265 270
Arg Arg Ser His Glu Val Asn Pro Ala Ala His Leu Thr Gly Ala Asn
275 280 285
Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu
290 295 300
Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val
305 310 315 320
Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly
325 330 335
Gly Val Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu
340 345 350
Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser
355 360 365
Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp
370 375 380
Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Lys
385 390 395 400
Val Val Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly
405 410 415
Thr Arg Ser Tyr Phe Gly Ala Phe Met Val
420 425
<210> 275
<211> 2022
<212> DNA
<213> 人工序列
<220>
<223> 编码含H75接头的单IL10-A2铰链::M0115融合蛋白的核苷酸序列
<400> 275
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacat caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacgg tggtggatcc 360
ggcggtaaga gcaaggccgt ggagcaggtg aagaatgcct ttaataagct ccaagagaaa 420
ggcatctaca aagccatgag tgagtttgac atcttcatca actacataga agcctacatt 480
acaatgaaga tacgaaacct cgagccccca cctcccccat gcccaccgtg cccagcacct 540
gaagccgcgg gtgcaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 600
atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 660
gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 720
gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 780
tggctgaatg gcaaggcgta cgcgtgcgcg gtctccaaca aagccctccc agcccccatc 840
gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 900
ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 960
tatccaagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1020
accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1080
gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1140
cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtcagaggca caacaattct 1200
tccctgaata caggaactca gatggcaggt cattctccga attctcaggt gcagctggtg 1260
cagtctgggg ctgaggtgaa gaagcctggg gcctcagtga aggtctcctg caaggcttct 1320
ggatacacct tcaccgacta caacatgaac tgggtgcgac aggcccctgg acaagggctt 1380
gagtggatgg gaaatattga tccttactat ggtggtacta gttacaatca gaagttcaag 1440
ggcagggtca ccatgaccag ggacacgtcc atcagcacag cctacatgga gctgagcagg 1500
ctgagatctg acgacacggc cgtgtattac tgtgcgagat gggactatag gtacgacgac 1560
gggagggctt actatgttat ggacttctgg ggccaaggga ccacggtcac cgtctcctca 1620
ggtggaggcg gttcaggcgg aggtggatcc ggcggtggcg gatcgggtgg cggcggatct 1680
gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 1740
atcaactgca agtccagcca gagtgtttta tacagctcca accagaagaa ctacttagct 1800
tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 1860
gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 1920
atcagcagcc tgcaggctga agatgtggca gtttattact gtcatcaata cctctactcg 1980
tggacgtttg gccaggggac caagctggag atcaaacggt aa 2022
<210> 276
<211> 673
<212> PRT
<213> 人工序列
<220>
<223> 含H75接头的单IL10-A2铰链::M0115融合蛋白
<400> 276
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu
115 120 125
Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
130 135 140
Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile
145 150 155 160
Thr Met Lys Ile Arg Asn Leu Glu Pro Pro Pro Pro Pro Cys Pro Pro
165 170 175
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
180 185 190
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
195 200 205
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
210 215 220
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
225 230 235 240
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
245 250 255
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
260 265 270
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
275 280 285
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
290 295 300
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
305 310 315 320
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
325 330 335
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
340 345 350
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
355 360 365
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
370 375 380
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gln Arg His Asn Asn Ser
385 390 395 400
Ser Leu Asn Thr Gly Thr Gln Met Ala Gly His Ser Pro Asn Ser Gln
405 410 415
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
420 425 430
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Asn
435 440 445
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
450 455 460
Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys
465 470 475 480
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met
485 490 495
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala
500 505 510
Arg Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met Asp
515 520 525
Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
530 535 540
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
545 550 555 560
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
565 570 575
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
580 585 590
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
595 600 605
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
610 615 620
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
625 630 635 640
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln
645 650 655
Tyr Leu Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
660 665 670
Arg
<210> 277
<211> 1185
<212> DNA
<213> 人工序列
<220>
<223> HVEM Ig合成分子
<400> 277
ctgccgtcct gcaaggagga cgagtaccca gtgggctccg agtgctgccc caagtgcagt 60
ccaggttatc gtgtgaagga ggcctgcggg gagctgacgg gcacagtgtg tgaaccctgc 120
cctccaggca cctacattgc ccacctcaat ggcctaagca agtgtctgca gtgccaaatg 180
tgtgacccag ccatgggcct gcgcgcgagc cggaactgct ccaggacaga gaacgccgtg 240
tgtggctgca gcccaggcca cttctgcatc gtccaggacg gggaccactg cgccgcgtgc 300
cgcgcttacg ccacctccag cccgggccag agggtgcaga agggaggcac cgagagtcag 360
gacaccctgt gtcagaactg ccccccgggg accttctctc ccaatgggac cctggaggaa 420
tgtcagcacc agaccaagtg cagctggctg gtgacgaagg ccggagctgg gaccagcagc 480
tcccacctcg agcccaaatc ttctgacaaa actcacacat gcccaccgtg cccagcacct 540
gaagccgcgg gtgcaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 600
atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 660
gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 720
gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 780
tggctgaatg gcaaggcgta cgcgtgcgcg gtctccaaca aagccctccc agcccccatc 840
gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 900
ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 960
tatccaagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1020
accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1080
gacaagagcc ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1140
cacaaccact acacgcagaa gagcctctcc ctgtctccgg gttaa 1185
<210> 278
<211> 394
<212> PRT
<213> 人工序列
<220>
<223> HVEM Ig合成分子
<400> 278
Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys
1 5 10 15
Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu
20 25 30
Thr Gly Thr Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His
35 40 45
Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala
50 55 60
Met Gly Leu Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val
65 70 75 80
Cys Gly Cys Ser Pro Gly His Phe Cys Ile Val Gln Asp Gly Asp His
85 90 95
Cys Ala Ala Cys Arg Ala Tyr Ala Thr Ser Ser Pro Gly Gln Arg Val
100 105 110
Gln Lys Gly Gly Thr Glu Ser Gln Asp Thr Leu Cys Gln Asn Cys Pro
115 120 125
Pro Gly Thr Phe Ser Pro Asn Gly Thr Leu Glu Glu Cys Gln His Gln
130 135 140
Thr Lys Cys Ser Trp Leu Val Thr Lys Ala Gly Ala Gly Thr Ser Ser
145 150 155 160
Ser His Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
165 170 175
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
180 185 190
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
195 200 205
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
210 215 220
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
225 230 235 240
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
245 250 255
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
260 265 270
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
275 280 285
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
290 295 300
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
305 310 315 320
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
325 330 335
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
340 345 350
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
355 360 365
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
370 375 380
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
385 390
<210> 279
<211> 1179
<212> DNA
<213> 人工序列
<220>
<223> IL10-Fc合成分子
<400> 279
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacat caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agccggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggttaa 1179
<210> 280
<211> 392
<212> PRT
<213> 人工序列
<220>
<223> IL10-Fc合成分子
<400> 280
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly
385 390
<210> 281
<211> 1179
<212> DNA
<213> 人工序列
<220>
<223> IL10 I87A-Fc合成分子
<400> 281
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacgc caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agccggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggttaa 1179
<210> 282
<211> 392
<212> PRT
<213> 人工序列
<220>
<223> IL10 I87A-Fc合成分子
<400> 282
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly
385 390
<210> 283
<211> 1197
<212> DNA
<213> 人工序列
<220>
<223> 单IL10-Fc合成分子
<400> 283
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacat caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacgg tggtggatcc 360
ggcggtaaga gcaaggccgt ggagcaggtg aagaatgcct ttaataagct ccaagagaaa 420
ggcatctaca aagccatgag tgagtttgac atcttcatca actacataga agcctacatg 480
acaatgaaga tacgaaacct cgagcccaaa tcttctgaca aaactcacac atgcccaccg 540
tgcccagcac ctgaagccgc gggtgcaccg tcagtcttcc tcttcccccc aaaacccaag 600
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 660
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 720
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 780
ctgcaccagg actggctgaa tggcaaggcg tacgcgtgcg cggtctccaa caaagccctc 840
ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 900
tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 960
gtcaaaggct tctatccaag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1020
aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1080
aagctcaccg tggacaagag ccggtggcag caggggaacg tcttctcatg ctccgtgatg 1140
catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggttaa 1197
<210> 284
<211> 398
<212> PRT
<213> 人工序列
<220>
<223> 单IL10-Fc合成分子
<400> 284
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu
115 120 125
Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
130 135 140
Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met
145 150 155 160
Thr Met Lys Ile Arg Asn Leu Glu Pro Lys Ser Ser Asp Lys Thr His
165 170 175
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val
180 185 190
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
195 200 205
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
210 215 220
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
225 230 235 240
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
245 250 255
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala
260 265 270
Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
275 280 285
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
290 295 300
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
305 310 315 320
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
325 330 335
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
340 345 350
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
355 360 365
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
370 375 380
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
385 390 395
<210> 285
<211> 1197
<212> DNA
<213> 人工序列
<220>
<223> 单IL10 I87A-Fc合成分子
<400> 285
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacgc caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacgg tggtggatcc 360
ggcggtaaga gcaaggccgt ggagcaggtg aagaatgcct ttaataagct ccaagagaaa 420
ggcatctaca aagccatgag tgagtttgac atcttcatca actacataga agcctacatt 480
acaatgaaga tacgaaacct cgagcccaaa tcttctgaca aaactcacac atgcccaccg 540
tgcccagcac ctgaagccgc gggtgcaccg tcagtcttcc tcttcccccc aaaacccaag 600
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 660
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 720
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 780
ctgcaccagg actggctgaa tggcaaggcg tacgcgtgcg cggtctccaa caaagccctc 840
ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 900
tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 960
gtcaaaggct tctatccaag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1020
aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1080
aagctcaccg tggacaagag ccggtggcag caggggaacg tcttctcatg ctccgtgatg 1140
catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggttaa 1197
<210> 286
<211> 398
<212> PRT
<213> 人工序列
<220>
<223> 单IL10 I87A-Fc合成分子
<400> 286
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu
115 120 125
Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
130 135 140
Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile
145 150 155 160
Thr Met Lys Ile Arg Asn Leu Glu Pro Lys Ser Ser Asp Lys Thr His
165 170 175
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val
180 185 190
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
195 200 205
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
210 215 220
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
225 230 235 240
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
245 250 255
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala
260 265 270
Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
275 280 285
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
290 295 300
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
305 310 315 320
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
325 330 335
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
340 345 350
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
355 360 365
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
370 375 380
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
385 390 395
<210> 287
<211> 1239
<212> DNA
<213> 人工序列
<220>
<223> Fc-IL10合成分子
<400> 287
gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 60
ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggcgt acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtcagaggc acaacaattc ttccctgaat 720
acaagaactc agaaagcacg tcattctccg aattctagcc caggccaggg cacccagtct 780
gagaacagct gcacccactt cccaggcaac ctgcctaaca tgcttcgaga tctccgagat 840
gccttcagca gagtgaagac tttctttcaa atgaaggatc agctggacaa cttgttgtta 900
aaggagtcct tgctggagga ctttaagggt tacctgggtt gccaagcctt gtctgagatg 960
atccagtttt acctggagga ggtgatgccc caagctgaga accaagaccc agacatcaag 1020
gcgcatgtga actccctggg ggagaacctg aagaccctca ggctgaggct acggcgctgt 1080
catcgatttc ttccctgtga aaacaagagc aaggccgtgg agcaggtgaa gaatgccttt 1140
aataagctcc aagagaaagg catctacaaa gccatgagtg agtttgacat cttcatcaac 1200
tacatagaag cctacatgac aatgaagata cgaaactaa 1239
<210> 288
<211> 412
<212> PRT
<213> 人工序列
<220>
<223> Fc-IL10合成分子
<400> 288
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn
225 230 235 240
Thr Arg Thr Gln Lys Ala Arg His Ser Pro Asn Ser Ser Pro Gly Gln
245 250 255
Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro
260 265 270
Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe
275 280 285
Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu
290 295 300
Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met
305 310 315 320
Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp
325 330 335
Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr
340 345 350
Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn
355 360 365
Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln
370 375 380
Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn
385 390 395 400
Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
405 410
<210> 289
<211> 1236
<212> DNA
<213> 人工序列
<220>
<223> Fc-IL10 I87A合成分子
<400> 289
gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 60
ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggcgt acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaactcat tattcaacca agaagttcaa 720
attcccttga ccgaaagtta cagcccgaat tctagcccag gccagggcac ccagtctgag 780
aacagctgca cccacttccc aggcaacctg cctaacatgc ttcgagatct ccgagatgcc 840
ttcagcagag tgaagacttt ctttcaaatg aaggatcagc tggacaactt gttgttaaag 900
gagtccttgc tggaggactt taagggttac ctgggttgcc aagccttgtc tgagatgatc 960
cagttttacc tggaggaggt gatgccccaa gctgagaacc aagacccaga cgccaaggcg 1020
catgtgaact ccctggggga gaacctgaag accctcaggc tgaggctacg gcgctgtcat 1080
cgatttcttc cctgtgaaaa caagagcaag gccgtggagc aggtgaagaa tgcctttaat 1140
aagctccaag agaaaggcat ctacaaagcc atgagtgagt ttgacatctt catcaactac 1200
atagaagcct acatgacaat gaagatacga aactaa 1236
<210> 290
<211> 411
<212> PRT
<213> 人工序列
<220>
<223> Fc-IL10 I87A合成分子
<400> 290
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln
225 230 235 240
Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Ser Pro Gly Gln Gly
245 250 255
Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn
260 265 270
Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe
275 280 285
Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu
290 295 300
Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile
305 310 315 320
Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro
325 330 335
Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu
340 345 350
Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys
355 360 365
Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu
370 375 380
Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr
385 390 395 400
Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
405 410
<210> 291
<211> 1254
<212> DNA
<213> 人工序列
<220>
<223> Fc-单IL10合成分子
<400> 291
gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 60
ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggcgt acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaactcat tattcaacca agaagttcaa 720
attcccttga ccgaaagtta cagcccgaat tctagcccag gccagggcac ccagtctgag 780
aacagctgca cccacttccc aggcaacctg cctaacatgc ttcgagatct ccgagatgcc 840
ttcagcagag tgaagacttt ctttcaaatg aaggatcagc tggacaactt gttgttaaag 900
gagtccttgc tggaggactt taagggttac ctgggttgcc aagccttgtc tgagatgatc 960
cagttttacc tggaggaggt gatgccccaa gctgagaacc aagacccaga catcaaggcg 1020
catgtgaact ccctggggga gaacctgaag accctcaggc tgaggctacg gcgctgtcat 1080
cgatttcttc cctgtgaaaa cggtggtgga tccggcggta agagcaaggc cgtggagcag 1140
gtgaagaatg cctttaataa gctccaagag aaaggcatct acaaagccat gagtgagttt 1200
gacatcttca tcaactacat agaagcctac atgacaatga agatacgaaa ctaa 1254
<210> 292
<211> 417
<212> PRT
<213> 人工序列
<220>
<223> Fc-单IL10合成分子
<400> 292
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln
225 230 235 240
Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Ser Pro Gly Gln Gly
245 250 255
Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn
260 265 270
Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe
275 280 285
Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu
290 295 300
Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile
305 310 315 320
Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro
325 330 335
Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu
340 345 350
Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Gly
355 360 365
Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala
370 375 380
Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe
385 390 395 400
Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg
405 410 415
Asn
<210> 293
<211> 1254
<212> DNA
<213> 人工序列
<220>
<223> Fc-单IL10 I87A合成分子
<400> 293
gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 60
ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggcgt acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaactcat tattcaacca agaagttcaa 720
attcccttga ccgaaagtta cagcccgaat tctagcccag gccagggcac ccagtctgag 780
aacagctgca cccacttccc aggcaacctg cctaacatgc ttcgagatct ccgagatgcc 840
ttcagcagag tgaagacttt ctttcaaatg aaggatcagc tggacaactt gttgttaaag 900
gagtccttgc tggaggactt taagggttac ctgggttgcc aagccttgtc tgagatgatc 960
cagttttacc tggaggaggt gatgccccaa gctgagaacc aagacccaga cgccaaggcg 1020
catgtgaact ccctggggga gaacctgaag accctcaggc tgaggctacg gcgctgtcat 1080
cgatttcttc cctgtgaaaa cggtggtgga tccggcggta agagcaaggc cgtggagcag 1140
gtgaagaatg cctttaataa gctccaagag aaaggcatct acaaagccat gagtgagttt 1200
gacatcttca tcaactacat agaagcctac atgacaatga agatacgaaa ctaa 1254
<210> 294
<211> 417
<212> PRT
<213> 人工序列
<220>
<223> Fc-单IL10 I87A合成分子
<400> 294
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln
225 230 235 240
Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Ser Pro Gly Gln Gly
245 250 255
Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn
260 265 270
Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe
275 280 285
Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu
290 295 300
Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile
305 310 315 320
Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro
325 330 335
Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu
340 345 350
Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Gly
355 360 365
Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala
370 375 380
Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe
385 390 395 400
Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg
405 410 415
Asn
<210> 295
<211> 1200
<212> DNA
<213> 人工序列
<220>
<223> 小鼠IL10-小鼠无效Fc合成分子
<400> 295
agcaggggcc agtacagccg ggaagacaat aactgcaccc acttcccagt cggccagagc 60
cacatgctcc tagagctgcg gactgccttc agccaggtga agactttctt tcaaacaaag 120
gaccagctgg acaacatact gctaaccgac tccttaatgc aggactttaa gggttacttg 180
ggttgccaag ccttatcgga aatgatccag ttttacctgg tagaagtgat gccccaggca 240
gagaagcatg gcccagaaat caaggagcat ttgaattccc tgggtgagaa gctgaagacc 300
ctcaggatgc ggctgaggcg ctgtcatcga tttctcccct gtgaaaataa gagcaaggca 360
gtggagcagg tgaagagtga ttttaataag ctccaagacc aaggtgtcta caaggccatg 420
aatgaatttg acatcttcat caactgcata gaagcataca tgatgatcaa aatgaaaagc 480
ctcgagccca gagtgcccat aacacagaac ccctgtcctc cactcaaaga gtgtccccca 540
tgcgcagctc cagacgcagc gggtgcgcca tccgtcttca tcttccctcc aaagatcaag 600
gatgtactca tgatctccct gagccccatg gtcacatgtg tggtggtgga tgtgagcgag 660
gatgacccag acgtccagat cagctggttt gtgaacaacg tggaagtaca cacagctcag 720
acacaaaccc atagagagga ttacaacagt actctccggg tggtcagtgc cctccccatc 780
cagcaccagg actggatgag tggcaaggcg ttcgcatgcg cggtcaacaa cagagccctc 840
ccatccccca tcgagaaaac catctcaaaa cccagagggc cagtaagagc tccacaggta 900
tatgtcttgc ctccaccagc agaagagatg actaagaaag agttcagtct gacctgcatg 960
atcgcaggct tcttacctgc cgaaattgct gtggactgga ccagcaatgg gcgtacagag 1020
caaaactaca agaacaccgc aacagtcctg gactctgatg gttcttactt catgtacagc 1080
aagctcagag tacaaaagag cacttgggaa agaggaagtc ttttcgcctg ctcagtggtc 1140
cacgagggtc tgcacaatca ccttacgact aagaccatct cccggtctct gggtaaataa 1200
<210> 296
<211> 399
<212> PRT
<213> 人工序列
<220>
<223> 小鼠IL10-小鼠无效Fc合成分子
<400> 296
Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His Phe Pro
1 5 10 15
Val Gly Gln Ser His Met Leu Leu Glu Leu Arg Thr Ala Phe Ser Gln
20 25 30
Val Lys Thr Phe Phe Gln Thr Lys Asp Gln Leu Asp Asn Ile Leu Leu
35 40 45
Thr Asp Ser Leu Met Gln Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Val Glu Val Met Pro Gln Ala
65 70 75 80
Glu Lys His Gly Pro Glu Ile Lys Glu His Leu Asn Ser Leu Gly Glu
85 90 95
Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Ser Asp Phe
115 120 125
Asn Lys Leu Gln Asp Gln Gly Val Tyr Lys Ala Met Asn Glu Phe Asp
130 135 140
Ile Phe Ile Asn Cys Ile Glu Ala Tyr Met Met Ile Lys Met Lys Ser
145 150 155 160
Leu Glu Pro Arg Val Pro Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys
165 170 175
Glu Cys Pro Pro Cys Ala Ala Pro Asp Ala Ala Gly Ala Pro Ser Val
180 185 190
Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser
195 200 205
Pro Met Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp
210 215 220
Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln
225 230 235 240
Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser
245 250 255
Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Ala Phe Ala
260 265 270
Cys Ala Val Asn Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile
275 280 285
Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro
290 295 300
Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys Met
305 310 315 320
Ile Ala Gly Phe Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn
325 330 335
Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser
340 345 350
Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr
355 360 365
Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val Val His Glu Gly Leu
370 375 380
His Asn His Leu Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly Lys
385 390 395
<210> 297
<211> 1095
<212> DNA
<213> 人工序列
<220>
<223> 小鼠CTLA4-小鼠无效2 Fc合成分子
<400> 297
gccatacagg tgacccaacc ttcagtggtg ttggctagca gccatggtgt cgccagcttt 60
ccatgtgaat attcaccatc acacaacact gatgaggtcc gggtgactgt gctgcggcag 120
acaaatgacc aaatgactga ggtctgtgcc acgacattca cagagaagaa tacagtgggc 180
ttcctagatt accccttctg cagtggtacc tttaatgaaa gcagagtgaa cctcaccatc 240
caaggactga gagctgttga cacgggactg tacctctgca aggtggaact catgtaccca 300
ccgccatact ttgtgggcat gggcaacggg acgcagattt atgtcattga tccagaacca 360
tgcccggatt ctgacctcga gcccagagtg cccataacac agaacccctg tcctccactc 420
aaagagtgtc ccccatgcgc agctccagac gcagcgggtg cgccatccgt cttcatcttc 480
cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 540
gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 600
gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 660
agtgccctcc ccatccagca ccaggactgg atgagtggca aggcgttcgc atgcgcggtc 720
aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 780
agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 840
agtctgacct gcatgatcgc aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 900
aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 960
tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1020
gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1080
tctctgggta aataa 1095
<210> 298
<211> 364
<212> PRT
<213> 人工序列
<220>
<223> 小鼠CTLA4-小鼠无效2 Fc合成分子
<400> 298
Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser His Gly
1 5 10 15
Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His Asn Thr Asp Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln Met Thr Glu Val
35 40 45
Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp Tyr
50 55 60
Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro
115 120 125
Arg Val Pro Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu Cys Pro
130 135 140
Pro Cys Ala Ala Pro Asp Ala Ala Gly Ala Pro Ser Val Phe Ile Phe
145 150 155 160
Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Met Val
165 170 175
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile
180 185 190
Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
195 200 205
His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro
210 215 220
Ile Gln His Gln Asp Trp Met Ser Gly Lys Ala Phe Ala Cys Ala Val
225 230 235 240
Asn Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro
245 250 255
Arg Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Ala
260 265 270
Glu Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys Met Ile Ala Gly
275 280 285
Phe Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn Gly Arg Thr
290 295 300
Glu Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser Asp Gly Ser
305 310 315 320
Tyr Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr Trp Glu Arg
325 330 335
Gly Ser Leu Phe Ala Cys Ser Val Val His Glu Gly Leu His Asn His
340 345 350
Leu Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly Lys
355 360
<210> 299
<211> 858
<212> DNA
<213> 人工序列
<220>
<223> NHP CD86-AFH合成分子
<400> 299
atgggactga gtaacattct ctttgtgatg gccttcctgc tctctggtgc tgctcccctg 60
aagattcagg cttatttcaa tgagactgca gacctgccat gccagtttgc aaactctcaa 120
aaccgaagcc tgagtgagct agtagtattt tggcagaacc aggaaaactt ggttctgaat 180
gaggtatact taggcaaaga gaaatttgac agcgttcatt ccaagtatat gggccgcaca 240
agttttgatc cggagagttg gaccctgagg cttcacaacc ttcagatcaa ggacaagggc 300
ttgtatcaat gtatcatcca ccacaaaagg cccacaggaa tgatccgcat ccaccagatg 360
aactctgaac tgtcagtgct tgctaacttc agtcaacctg aaatagtccc aatttctaat 420
ataacagaaa atatgtacat aaatttgacc tgctcatcta tacacggtta cccagaacct 480
gagaagatga gtgttttgct aagaaccaag aactcaacta tcgagtatga tggtgttatg 540
cagaaatctc aagataatgt cacagaactg tacgacgttt ccatcagctt gtctgtttca 600
ttccctgatg ttacgagcaa catgaccatc ttctgtgttc tggaaactga caagacacag 660
cttttatcct cacctttctc tatagagctt gaggaccctc agcctccccc agactcctcg 720
agcctgaacg acatcttcga ggctcagaaa atcgaatggc acgaagatta caaggatgac 780
gacgataagg attacaagga tgacgacgat aaggattaca aggatgacga cgataagcat 840
catcatcatc atcactga 858
<210> 300
<211> 285
<212> PRT
<213> 人工序列
<220>
<223> NHP CD86-AFH合成分子
<400> 300
Asn Gly Leu Ser Asn Ile Leu Phe Val Met Ala Phe Leu Leu Ser Gly
1 5 10 15
Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe Asn Glu Thr Ala Asp Leu
20 25 30
Pro Cys Gln Phe Ala Asn Ser Gln Asn Arg Ser Leu Ser Glu Leu Val
35 40 45
Val Phe Trp Gln Asn Gln Glu Asn Leu Val Leu Asn Glu Val Tyr Leu
50 55 60
Gly Lys Glu Lys Phe Asp Ser Val His Ser Lys Tyr Met Gly Arg Thr
65 70 75 80
Ser Phe Asp Pro Glu Ser Trp Thr Leu Arg Leu His Asn Leu Gln Ile
85 90 95
Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile His His Lys Arg Pro Thr
100 105 110
Gly Met Ile Arg Ile His Gln Met Asn Ser Glu Leu Ser Val Leu Ala
115 120 125
Asn Phe Ser Gln Pro Glu Ile Val Pro Ile Ser Asn Ile Thr Glu Asn
130 135 140
Met Tyr Ile Asn Leu Thr Cys Ser Ser Ile His Gly Tyr Pro Glu Pro
145 150 155 160
Glu Lys Met Ser Val Leu Leu Arg Thr Lys Asn Ser Thr Ile Glu Tyr
165 170 175
Asp Gly Val Met Gln Lys Ser Gln Asp Asn Val Thr Glu Leu Tyr Asp
180 185 190
Val Ser Ile Ser Leu Ser Val Ser Phe Pro Asp Val Thr Ser Asn Met
195 200 205
Thr Ile Phe Cys Val Leu Glu Thr Asp Lys Thr Gln Leu Leu Ser Ser
210 215 220
Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln Pro Pro Pro Asp Ser Ser
225 230 235 240
Ser Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp
245 250 255
Tyr Lys Asp Asp Asp Asp Lys Asp Tyr Lys Asp Asp Asp Asp Lys Asp
260 265 270
Tyr Lys Asp Asp Asp Asp Lys His His His His His His
275 280 285
<210> 301
<211> 1629
<212> DNA
<213> 人工序列
<220>
<223> 编码含H69接头的CTLA4::IL10融合蛋白的核苷酸序列
<400> 301
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtga gcagaagctg 1080
agcaacatgg agaacagact gaagcccttc ttcacaagcg gctcagcaga cacctccccg 1140
aattctagcc caggccaggg cacccagtct gagaacagct gcacccactt cccaggcaac 1200
ctgcctaaca tgcttcgaga tctccgagat gccttcagca gagtgaagac tttctttcaa 1260
atgaaggatc agctggacaa cttgttgtta aaggagtcct tgctggagga ctttaagggt 1320
tacctgggtt gccaagcctt gtctgagatg atccagtttt acctggagga ggtgatgccc 1380
caagctgaga accaagaccc agacatcaag gcgcatgtga actccctggg ggagaacctg 1440
aagaccctca ggctgaggct acggcgctgt catcgatttc ttccctgtga aaacaagagc 1500
aaggccgtgg agcaggtgaa gaatgccttt aataagctcc aagagaaagg catctacaaa 1560
gccatgagtg agtttgacat cttcatcaac tacatagaag cctacatgac aatgaagata 1620
cgaaactaa 1629
<210> 302
<211> 542
<212> PRT
<213> 人工序列
<220>
<223> 含69接头的CTLA4::IL10融合蛋白
<400> 302
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Glu Gln Lys Leu Ser Asn Met Glu Asn Arg Leu Lys
355 360 365
Pro Phe Phe Thr Ser Gly Ser Ala Asp Thr Ser Pro Asn Ser Ser Pro
370 375 380
Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn
385 390 395 400
Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys
405 410 415
Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu
420 425 430
Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser
435 440 445
Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn
450 455 460
Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu
465 470 475 480
Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys
485 490 495
Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys
500 505 510
Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe
515 520 525
Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
530 535 540
<210> 303
<211> 1473
<212> DNA
<213> 人工序列
<220>
<223> FUN1 SMIP合成分子
<400> 303
gaggtccaac tgcagcagtc tggacctgag ctggagaagc ctggcgcttc agtgaagata 60
tcctgcaagg cttctggtta ctcattcact gactacaaca tgaactgggt gaagcagagc 120
aatggaaaga gccttgagtg gattggaaat attgatcctt actatggtgg tactagttac 180
aatcagaagt tcaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240
atgcagctca acagcctgac atctgaagac tctgcagtct atttctgtgc aagatgggac 300
tataggtacg acgacgggag ggcttactat gttatggact tctggggtca aggaacctca 360
gtcaccgtct cctcaggggg tggaggctct ggtggcggtg gctctggcgg aggtggatcc 420
ggtggcggcg gatctgagct ccagatgacc cagtctccat catctctggc tgcgtctgca 480
ggagaaaagg tcactatgag ctgtaagtcc agtcaaagtg ttttatacag ttcaaatcag 540
aagaactact tggcctggta ccagcagaaa ccagggcagt ctcctaaact gctgatctac 600
tgggcatcca ctagggaatc tggtgtccct gatcgcttca caggcagtgg atctgggaca 660
cattttactc tgaccgtcag cagtgtgcaa gctgaagacc tggcagttta ttactgtcat 720
caatacctct actcgtggac gttcggtgga ggcaccaacc tggaaatcaa acggctcgag 780
cccaaatctt ctgacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgcgggt 840
gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080
aaggcgtacg cgtgcgcggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tccaagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagccgg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt taa 1473
<210> 304
<211> 490
<212> PRT
<213> 人工序列
<220>
<223> FUN1 SMIP合成分子
<400> 304
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr
20 25 30
Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
100 105 110
Asp Phe Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Glu Leu Gln Met Thr Gln Ser Pro Ser Ser Leu Ala Ala Ser Ala
145 150 155 160
Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr
165 170 175
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
195 200 205
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr His Phe Thr Leu
210 215 220
Thr Val Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys His
225 230 235 240
Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile
245 250 255
Lys Arg Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
260 265 270
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
340 345 350
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
355 360 365
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
485 490
<210> 305
<211> 125
<212> PRT
<213> 人工序列
<220>
<223> FUN1重链
<400> 305
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr
20 25 30
Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
100 105 110
Asp Phe Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120 125
<210> 306
<211> 113
<212> PRT
<213> 人工序列
<220>
<223> FUN1轻链
<400> 306
Glu Leu Gln Met Thr Gln Ser Pro Ser Ser Leu Ala Ala Ser Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr
65 70 75 80
Val Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys His Gln
85 90 95
Tyr Leu Tyr Ser Trp Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys
100 105 110
Arg
<210> 307
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> FUN1 scFV 接头
<400> 307
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 308
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> FUN1 HCDR1
<400> 308
Asp Tyr Asn Met Asn
1 5
<210> 309
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> FUN1 HCDR2
<400> 309
Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly
1 5 10 15
<210> 310
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> FUN1 HCDR3
<400> 310
Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met Asp Phe
1 5 10 15
<210> 311
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> FUN1 LCDR1
<400> 311
Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
<210> 312
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> FUN1 LCDR2
<400> 312
Trp Ala Ser Thr Arg Glu Ser
1 5
<210> 313
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> FUN1 LCDR3
<400> 313
His Gln Tyr Leu Tyr Ser Trp Thr
1 5
<210> 314
<211> 1530
<212> DNA
<213> 人工序列
<220>
<223> FUN1 PIMS合成分子
<400> 314
gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 60
ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggcgt acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaactcat tattcaacca agaagttcaa 720
attcccttga ccgaaagtta cagcccgaat tctgaggtcc aactgcagca gtctggacct 780
gagctggaga agcctggcgc ttcagtgaag atatcctgca aggcttctgg ttactcattc 840
actgactaca acatgaactg ggtgaagcag agcaatggaa agagccttga gtggattgga 900
aatattgatc cttactatgg tggtactagt tacaatcaga agttcaaggg caaggccaca 960
ttgactgtag acaaatcctc cagcacagcc tacatgcagc tcaacagcct gacatctgaa 1020
gactctgcag tctatttctg tgcaagatgg gactataggt acgacgacgg gagggcttac 1080
tatgttatgg acttctgggg tcaaggaacc tcagtcaccg tctcctcagg gggtggaggc 1140
tctggtggcg gtggctctgg cggaggtgga tccggtggcg gcggatctga gctccagatg 1200
acccagtctc catcatctct ggctgcgtct gcaggagaaa aggtcactat gagctgtaag 1260
tccagtcaaa gtgttttata cagttcaaat cagaagaact acttggcctg gtaccagcag 1320
aaaccagggc agtctcctaa actgctgatc tactgggcat ccactaggga atctggtgtc 1380
cctgatcgct tcacaggcag tggatctggg acacatttta ctctgaccgt cagcagtgtg 1440
caagctgaag acctggcagt ttattactgt catcaatacc tctactcgtg gacgttcggt 1500
ggaggcacca acctggaaat caaacggtaa 1530
<210> 315
<211> 509
<212> PRT
<213> 人工序列
<220>
<223> FUN1 PIMS合成分子
<400> 315
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln
225 230 235 240
Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Glu Val Gln Leu Gln
245 250 255
Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser Val Lys Ile Ser
260 265 270
Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Asn Met Asn Trp Val
275 280 285
Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile Gly Asn Ile Asp Pro
290 295 300
Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr
305 310 315 320
Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Asn Ser
325 330 335
Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Trp Asp Tyr
340 345 350
Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met Asp Phe Trp Gly Gln
355 360 365
Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
370 375 380
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Met
385 390 395 400
Thr Gln Ser Pro Ser Ser Leu Ala Ala Ser Ala Gly Glu Lys Val Thr
405 410 415
Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Gln Lys
420 425 430
Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
435 440 445
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe
450 455 460
Thr Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr Val Ser Ser Val
465 470 475 480
Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys His Gln Tyr Leu Tyr Ser
485 490 495
Trp Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys Arg
500 505
<210> 316
<211> 1443
<212> DNA
<213> 人工序列
<220>
<223> 3D1 SMIP合成分子
<400> 316
caggtccagc tgcagcagtc tgggcctgag ctggtgaggc ctggggaatc agtgaagatt 60
tcctgcaagg gttccggcta cacattcact gattatgcta tacagtgggt gaagcagagt 120
catgcaaaga gtctagagtg gattggagtt attaatattt actatgataa tacaaactac 180
aaccagaagt ttaagggcaa ggccacaatg actgtagaca aatcctccag cacagcctat 240
atggaacttg ccagattgac atctgaggat tctgccatct attactgtgc aagagcggcc 300
tggtatatgg actactgggg tcaaggaacc tcagtcaccg tctcctcagg gggtggaggc 360
tctggtggcg gtggctctgg cggaggtgga tccggtggcg gcggatctga cattgtgctg 420
tcacagtctc catcctccct ggctgtgtca gcaggagaga aggtcactat gagctgcaaa 480
tccagtcaga gtctgctcaa cagtagaacc cgagagaact acttggcttg gtaccagcag 540
aaaccagggc agtctcctaa actgctgatc tactgggcat ccactaggga atctggggtc 600
cctgatcgct tcacaggcag tggatctggg acagatttca ctctcaccat cagcagtgtg 660
caggctgaag acctggcagt ttattactgc acgcaatctt ataatcttta cacgttcgga 720
ggggggacca agctggaaat aaaactcgag cccaaatctt ctgacaaaac tcacacatgc 780
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 840
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 900
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 960
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 1020
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 1080
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1140
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 1200
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 1260
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1320
tacagcaagc tcaccgtgga caagagccgg tggcagcagg ggaacgtctt ctcatgctcc 1380
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1440
taa 1443
<210> 317
<211> 480
<212> PRT
<213> 人工序列
<220>
<223> 3D1 SMIP合成分子
<400> 317
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Glu
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile Gln Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile
35 40 45
Gly Val Ile Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Ala Ala Trp Tyr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Ser Gln Ser Pro
130 135 140
Ser Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys
145 150 155 160
Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr Leu Ala
165 170 175
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp
180 185 190
Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
195 200 205
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp
210 215 220
Leu Ala Val Tyr Tyr Cys Thr Gln Ser Tyr Asn Leu Tyr Thr Phe Gly
225 230 235 240
Gly Gly Thr Lys Leu Glu Ile Lys Leu Glu Pro Lys Ser Ser Asp Lys
245 250 255
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
260 265 270
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
275 280 285
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
290 295 300
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
305 310 315 320
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
325 330 335
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala
340 345 350
Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
355 360 365
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
370 375 380
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
385 390 395 400
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
405 410 415
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
420 425 430
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
435 440 445
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
450 455 460
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
465 470 475 480
<210> 318
<211> 116
<212> PRT
<213> 人工序列
<220>
<223> 3D1重链
<400> 318
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Glu
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile Gln Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile
35 40 45
Gly Val Ile Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Ala Ala Trp Tyr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser
115
<210> 319
<211> 112
<212> PRT
<213> 人工序列
<220>
<223> 3D1轻链
<400> 319
Asp Ile Val Leu Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Arg Thr Arg Glu Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Thr Gln
85 90 95
Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 320
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 3D1scFv 接头
<400> 320
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 321
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 3D1 HCDR1
<400> 321
Asp Tyr Ala Ile Gln
1 5
<210> 322
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 3D1 HCDR2
<400> 322
Val Ile Asn Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 323
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 3D1 HCDR3
<400> 323
Ala Ala Trp Tyr Met Asp Tyr
1 5
<210> 324
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 3D1 LCDR1
<400> 324
Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Glu Asn Tyr Leu
1 5 10 15
Ala
<210> 325
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 3D1 LCDR2
<400> 325
Trp Ala Ser Thr Arg Glu Ser
1 5
<210> 326
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 3D1 LCDR3
<400> 326
Thr Gln Ser Tyr Asn Leu Tyr Thr
1 5
<210> 327
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 3D1 PIMS合成分子
<400> 327
gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 60
ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggcgt acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaactcat tattcaacca agaagttcaa 720
attcccttga ccgaaagtta cagcccgaat tctcaggtcc agctgcagca gtctgggcct 780
gagctggtga ggcctgggga atcagtgaag atttcctgca agggttccgg ctacacattc 840
actgattatg ctatacagtg ggtgaagcag agtcatgcaa agagtctaga gtggattgga 900
gttattaata tttactatga taatacaaac tacaaccaga agtttaaggg caaggccaca 960
atgactgtag acaaatcctc cagcacagcc tatatggaac ttgccagatt gacatctgag 1020
gattctgcca tctattactg tgcaagagcg gcctggtata tggactactg gggtcaagga 1080
acctcagtca ccgtctcctc agggggtgga ggctctggtg gcggtggctc tggcggaggt 1140
ggatccggtg gcggcggatc tgacattgtg ctgtcacagt ctccatcctc cctggctgtg 1200
tcagcaggag agaaggtcac tatgagctgc aaatccagtc agagtctgct caacagtaga 1260
acccgagaga actacttggc ttggtaccag cagaaaccag ggcagtctcc taaactgctg 1320
atctactggg catccactag ggaatctggg gtccctgatc gcttcacagg cagtggatct 1380
gggacagatt tcactctcac catcagcagt gtgcaggctg aagacctggc agtttattac 1440
tgcacgcaat cttataatct ttacacgttc ggagggggga ccaagctgga aataaaataa 1500
<210> 328
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 3D1 PIMS合成分子
<400> 328
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln
225 230 235 240
Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Gln Val Gln Leu Gln
245 250 255
Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Glu Ser Val Lys Ile Ser
260 265 270
Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala Ile Gln Trp Val
275 280 285
Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile Gly Val Ile Asn Ile
290 295 300
Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr
305 310 315 320
Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ala Arg
325 330 335
Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg Ala Ala Trp
340 345 350
Tyr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly
355 360 365
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
370 375 380
Gly Gly Ser Asp Ile Val Leu Ser Gln Ser Pro Ser Ser Leu Ala Val
385 390 395 400
Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu
405 410 415
Leu Asn Ser Arg Thr Arg Glu Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
420 425 430
Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu
435 440 445
Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
450 455 460
Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr
465 470 475 480
Cys Thr Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu
485 490 495
Glu Ile Lys
<210> 329
<211> 2016
<212> DNA
<213> 人工序列
<220>
<223> 编码含H75接头的M0115::IL10 I87A融合蛋白的核苷酸序列
<400> 329
caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60
tcctgcaagg cttctggata caccttcacc gactacaaca tgaactgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaaat attgatcctt actatggtgg tactagttac 180
aatcagaagt tcaagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240
atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagatgggac 300
tataggtacg acgacgggag ggcttactat gttatggact tctggggcca agggaccacg 360
gtcaccgtct cctcaggtgg aggcggttca ggcggaggtg gatccggcgg tggcggatcg 420
ggtggcggcg gatctgacat cgtgatgacc cagtctccag actccctggc tgtgtctctg 480
ggcgagaggg ccaccatcaa ctgcaagtcc agccagagtg ttttatacag ctccaaccag 540
aagaactact tagcttggta ccagcagaaa ccaggacagc ctcctaagct gctcatttac 600
tgggcatcta cccgggaatc cggggtccct gaccgattca gtggcagcgg gtctgggaca 660
gatttcactc tcaccatcag cagcctgcag gctgaagatg tggcagttta ttactgtcat 720
caatacctct actcgtggac gtttggccag gggaccaagc tggagatcaa acggctcgag 780
cccaaatctt ctgacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgcgggt 840
gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080
aaggcgtacg cgtgcgcggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tccaagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt cagaggcaca acaattcttc cctgaataca 1500
ggaactcaga tggcaggtca ttctccgaat tctagcccag gccagggcac ccagtctgag 1560
aacagctgca cccacttccc aggcaacctg cctaacatgc ttcgagatct ccgagatgcc 1620
ttcagcagag tgaagacttt ctttcaaatg aaggatcagc tggacaactt gttgttaaag 1680
gagtccttgc tggaggactt taagggttac ctgggttgcc aagccttgtc tgagatgatc 1740
cagttttacc tggaggaggt gatgccccaa gctgagaacc aagacccaga cgccaaggcg 1800
catgtgaact ccctggggga gaacctgaag accctcaggc tgaggctacg gcgctgtcat 1860
cgatttcttc cctgtgaaaa caagagcaag gccgtggagc aggtgaagaa tgcctttaat 1920
aagctccaag agaaaggcat ctacaaagcc atgagtgagt ttgacatctt catcaactac 1980
atagaagcct acatgacaat gaagatacga aactaa 2016
<210> 330
<211> 671
<212> PRT
<213> 人工序列
<220>
<223> 含H75接头的M0115::IL10 I87A融合蛋白
<400> 330
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met
100 105 110
Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
145 150 155 160
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
165 170 175
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
180 185 190
Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
195 200 205
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
210 215 220
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His
225 230 235 240
Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
245 250 255
Lys Arg Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
260 265 270
Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
340 345 350
Leu His Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser
355 360 365
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gln Arg His Asn Asn Ser
485 490 495
Ser Leu Asn Thr Gly Thr Gln Met Ala Gly His Ser Pro Asn Ser Ser
500 505 510
Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly
515 520 525
Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val
530 535 540
Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys
545 550 555 560
Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu
565 570 575
Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu
580 585 590
Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu Asn
595 600 605
Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro
610 615 620
Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn
625 630 635 640
Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile
645 650 655
Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
660 665 670
<210> 331
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> GL1 PIMS合成分子
<400> 331
gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 60
ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggcgt acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaactcat tattcaacca agaagttcaa 720
attcccttga ccgaaagtta cagcccgaat tctgaggtgc agctggtgga gtctgctgga 780
ggtttagtgc agcctggaag gtccctaaaa ctctcctgtg cagcctcagg attcactttc 840
agtgactatt acatggcctg ggtccgccag gctccaacga aggggctgga gtgggtcgca 900
accattagtc atgacggtag tagtacttac tatcgagact ccgtgaaggg ccgattcaca 960
atctccagag ataatgcaaa aaccatccta tacctgcaaa tggacagtct gaggtctgag 1020
gacacggcca cttatttctg tgcaagacag aatattatgg attattgggg ccaaggagtc 1080
atggtcacag tctcctcagg gggtggaggc tctggtggcg gtggctctgg cggaggtgga 1140
tccggtggcg gcggatctga cattgtgatg acccagactc catcctccca ggctgtgtca 1200
gcaggggaga aggtcactat gagctgcaag tccagtcaga gtcttttgta cagtagagac 1260
cagagtaact acttggcctg gtaccagcag aaacctgggc agtctcctaa actacttatc 1320
tacttggcat ccactaggga atcaggggtc cctgatcgct tcataggcag tggctctggg 1380
acagacttca ctctgaccat cagcagtgtg caggctgaag atctggcaga ttattactgt 1440
cagcagcatt acaactatcc gctcacgttc ggttctggga ccaagctaga aatcaaataa 1500
<210> 332
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> GL1 PIMS合成分子
<400> 332
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln
225 230 235 240
Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Glu Val Gln Leu Val
245 250 255
Glu Ser Ala Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Lys Leu Ser
260 265 270
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Ala Trp Val
275 280 285
Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val Ala Thr Ile Ser His
290 295 300
Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr
305 310 315 320
Ile Ser Arg Asp Asn Ala Lys Thr Ile Leu Tyr Leu Gln Met Asp Ser
325 330 335
Leu Arg Ser Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gln Asn Ile
340 345 350
Met Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser Gly Gly
355 360 365
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
370 375 380
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Ser Ser Gln Ala Val Ser
385 390 395 400
Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu
405 410 415
Tyr Ser Arg Asp Gln Ser Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
420 425 430
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Leu Ala Ser Thr Arg Glu Ser
435 440 445
Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr
450 455 460
Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Tyr Cys
465 470 475 480
Gln Gln His Tyr Asn Tyr Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu
485 490 495
Glu Ile Lys
<210> 333
<211> 1986
<212> DNA
<213> 人工序列
<220>
<223> 编码含H75接头的GL1::IL10 I87A融合蛋白的核苷酸序列
<400> 333
gaggtgcagc tggtggagtc tgctggaggt ttagtgcagc ctggaaggtc cctaaaactc 60
tcctgtgcag cctcaggatt cactttcagt gactattaca tggcctgggt ccgccaggct 120
ccaacgaagg ggctggagtg ggtcgcaacc attagtcatg acggtagtag tacttactat 180
cgagactccg tgaagggccg attcacaatc tccagagata atgcaaaaac catcctatac 240
ctgcaaatgg acagtctgag gtctgaggac acggccactt atttctgtgc aagacagaat 300
attatggatt attggggcca aggagtcatg gtcacagtct cctcaggggg tggaggctct 360
ggtggcggtg gctctggcgg aggtggatcc ggtggcggcg gatctgacat tgtgatgacc 420
cagactccat cctcccaggc tgtgtcagca ggggagaagg tcactatgag ctgcaagtcc 480
agtcagagtc ttttgtacag tagagaccag agtaactact tggcctggta ccagcagaaa 540
cctgggcagt ctcctaaact acttatctac ttggcatcca ctagggaatc aggggtccct 600
gatcgcttca taggcagtgg ctctgggaca gacttcactc tgaccatcag cagtgtgcag 660
gctgaagatc tggcagatta ttactgtcag cagcattaca actatccgct cacgttcggt 720
tctgggacca agctagaaat caaactcgag cccaaatctt ctgacaaaac tcacacatgc 780
ccaccgtgcc cagcacctga agccgcgggt gcaccgtcag tcttcctctt ccccccaaaa 840
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 900
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 960
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 1020
accgtcctgc accaggactg gctgaatggc aaggcgtacg cgtgcgcggt ctccaacaaa 1080
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1140
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 1200
tgcctggtca aaggcttcta tccaagcgac atcgccgtgg agtgggagag caatgggcag 1260
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1320
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1380
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1440
cagaggcaca acaattcttc cctgaataca ggaactcaga tggcaggtca ttctccgaat 1500
tctagcccag gccagggcac ccagtctgag aacagctgca cccacttccc aggcaacctg 1560
cctaacatgc ttcgagatct ccgagatgcc ttcagcagag tgaagacttt ctttcaaatg 1620
aaggatcagc tggacaactt gttgttaaag gagtccttgc tggaggactt taagggttac 1680
ctgggttgcc aagccttgtc tgagatgatc cagttttacc tggaggaggt gatgccccaa 1740
gctgagaacc aagacccaga cgccaaggcg catgtgaact ccctggggga gaacctgaag 1800
accctcaggc tgaggctacg gcgctgtcat cgatttcttc cctgtgaaaa caagagcaag 1860
gccgtggagc aggtgaagaa tgcctttaat aagctccaag agaaaggcat ctacaaagcc 1920
atgagtgagt ttgacatctt catcaactac atagaagcct acatgacaat gaagatacga 1980
aactaa 1986
<210> 334
<211> 661
<212> PRT
<213> 人工序列
<220>
<223> 含H75接头的GL1::IL10 I87A融合蛋白
<400> 334
Glu Val Gln Leu Val Glu Ser Ala Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser His Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Ile Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Gln Asn Ile Met Asp Tyr Trp Gly Gln Gly Val Met Val Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro Ser
130 135 140
Ser Gln Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser
145 150 155 160
Ser Gln Ser Leu Leu Tyr Ser Arg Asp Gln Ser Asn Tyr Leu Ala Trp
165 170 175
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Leu Ala
180 185 190
Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser
195 200 205
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu
210 215 220
Ala Asp Tyr Tyr Cys Gln Gln His Tyr Asn Tyr Pro Leu Thr Phe Gly
225 230 235 240
Ser Gly Thr Lys Leu Glu Ile Lys Leu Glu Pro Lys Ser Ser Asp Lys
245 250 255
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
260 265 270
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
275 280 285
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
290 295 300
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
305 310 315 320
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
325 330 335
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Ala
340 345 350
Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
355 360 365
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
370 375 380
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
385 390 395 400
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
405 410 415
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
420 425 430
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
435 440 445
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
450 455 460
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
465 470 475 480
Gln Arg His Asn Asn Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Gly
485 490 495
His Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser
500 505 510
Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg
515 520 525
Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu
530 535 540
Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr
545 550 555 560
Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu
565 570 575
Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val
580 585 590
Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg
595 600 605
Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln
610 615 620
Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala
625 630 635 640
Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr
645 650 655
Met Lys Ile Arg Asn
660
<210> 335
<211> 1737
<212> DNA
<213> 人工序列
<220>
<223> 编码含H65接头的CTLA4::PDL2融合蛋白的核苷酸序列
<400> 335
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtca gaggcacaac 1080
aattcttccc tgaatacaag aactcagaaa gcacgtcatt ctccgaattc tttattcaca 1140
gtgacagtcc ctaaggaact gtacataata gagcatggca gcaatgtgac cctggaatgc 1200
aactttgaca ctggaagtca tgtgaacctt ggagcaataa cagccagttt gcaaaaggtg 1260
gaaaatgata catccccaca ccgtgaaaga gccactttgc tggaggagca gctgccccta 1320
gggaaggcct cgttccacat acctcaagtc caagtgaggg acgaaggaca gtaccaatgc 1380
ataatcatct atggggtcgc ctgggactac aagtacctga ccctgaaagt caaagcttcc 1440
tacaggaaaa taaacactca catcctaaag gttccagaaa cagatgaggt agagctcacc 1500
tgccaggcta caggttatcc tctggcagaa gtatcctggc caaacgtcag cgttcctgcc 1560
aacaccagcc actccaggac ccctgaaggc ctctaccagg tcaccagtgt tctgcgccta 1620
aagccacccc ctggcagaaa cttcagctgt gtgttctgga atactcacgt gagggaactt 1680
actttggcca gcattgacct tcaaagtcag atggaaccca ggacccatcc aacttaa 1737
<210> 336
<211> 578
<212> PRT
<213> 人工序列
<220>
<223> 含H65接头的CTLA4::PDL2融合蛋白
<400> 336
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr
355 360 365
Gln Lys Ala Arg His Ser Pro Asn Ser Leu Phe Thr Val Thr Val Pro
370 375 380
Lys Glu Leu Tyr Ile Ile Glu His Gly Ser Asn Val Thr Leu Glu Cys
385 390 395 400
Asn Phe Asp Thr Gly Ser His Val Asn Leu Gly Ala Ile Thr Ala Ser
405 410 415
Leu Gln Lys Val Glu Asn Asp Thr Ser Pro His Arg Glu Arg Ala Thr
420 425 430
Leu Leu Glu Glu Gln Leu Pro Leu Gly Lys Ala Ser Phe His Ile Pro
435 440 445
Gln Val Gln Val Arg Asp Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr
450 455 460
Gly Val Ala Trp Asp Tyr Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser
465 470 475 480
Tyr Arg Lys Ile Asn Thr His Ile Leu Lys Val Pro Glu Thr Asp Glu
485 490 495
Val Glu Leu Thr Cys Gln Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser
500 505 510
Trp Pro Asn Val Ser Val Pro Ala Asn Thr Ser His Ser Arg Thr Pro
515 520 525
Glu Gly Leu Tyr Gln Val Thr Ser Val Leu Arg Leu Lys Pro Pro Pro
530 535 540
Gly Arg Asn Phe Ser Cys Val Phe Trp Asn Thr His Val Arg Glu Leu
545 550 555 560
Thr Leu Ala Ser Ile Asp Leu Gln Ser Gln Met Glu Pro Arg Thr His
565 570 575
Pro Thr
<210> 337
<211> 1551
<212> DNA
<213> 人工序列
<220>
<223> 编码含H65接头的CTLA4::GITR融合蛋白的核苷酸序列
<400> 337
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtca gaggcacaac 1080
aattcttccc tgaatacaag aactcagaaa gcacgtcatt ctccgaattc tcagcgcccc 1140
accgggggtc ccgggtgcgg ccctgggcgc ctcctgcttg ggacgggaac ggacgcgcgc 1200
tgctgccggg ttcacacgac gcgctgctgc cgcgattacc cgggcgagga gtgctgttcc 1260
gagtgggact gcatgtgtgt ccagcctgaa ttccactgcg gagacccttg ctgcacgacc 1320
tgccggcacc acccttgtcc cccaggccag ggggtacagt cccaggggaa attcagtttt 1380
ggcttccagt gtatcgactg tgcctcgggg accttctccg ggggccacga aggccactgc 1440
aaaccttgga cagactgcac ccagttcggg tttctcactg tgttccctgg gaacaagacc 1500
cacaacgctg tgtgcgtccc agggtccccc ccggcagagc cgcttgggta a 1551
<210> 338
<211> 516
<212> PRT
<213> 人工序列
<220>
<223> 含HC65接头的CTLA4::GITR融合蛋白
<400> 338
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr
355 360 365
Gln Lys Ala Arg His Ser Pro Asn Ser Gln Arg Pro Thr Gly Gly Pro
370 375 380
Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly Thr Asp Ala Arg
385 390 395 400
Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp Tyr Pro Gly Glu
405 410 415
Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln Pro Glu Phe His
420 425 430
Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His Pro Cys Pro Pro
435 440 445
Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe Gly Phe Gln Cys
450 455 460
Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His Glu Gly His Cys
465 470 475 480
Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu Thr Val Phe Pro
485 490 495
Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly Ser Pro Pro Ala
500 505 510
Glu Pro Leu Gly
515
<210> 339
<211> 1437
<212> DNA
<213> 人工序列
<220>
<223> 编码含H65接头的CTLA4::TGFβR1融合蛋白的核苷酸序列
<400> 339
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtca gaggcacaac 1080
aattcttccc tgaatacaag aactcagaaa gcacgtcatt ctccgaattc tgcggcgctg 1140
ctcccggggg cgacggcgtt acagtgtttc tgccacctct gtacaaaaga caattttact 1200
tgtgtgacag atgggctctg ctttgtctct gtcacagaga ccacagacaa agttatacac 1260
aacagcatgt gtatagctga aattgactta attcctcgag ataggccgtt tgtatgtgca 1320
ccctcttcaa aaactgggtc tgtgactaca acatattgct gcaatcagga ccattgcaat 1380
aaaatagaac ttccaactac tgtaaagtca tcacctggcc ttggtcctgt ggaataa 1437
<210> 340
<211> 478
<212> PRT
<213> 人工序列
<220>
<223> 含H65接头的CTLA4::TGFβR1融合蛋白
<400> 340
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr
355 360 365
Gln Lys Ala Arg His Ser Pro Asn Ser Ala Ala Leu Leu Pro Gly Ala
370 375 380
Thr Ala Leu Gln Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr
385 390 395 400
Cys Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp
405 410 415
Lys Val Ile His Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro
420 425 430
Arg Asp Arg Pro Phe Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val
435 440 445
Thr Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu
450 455 460
Pro Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu
465 470 475
<210> 341
<211> 798
<212> DNA
<213> 人工序列
<220>
<223> 人CD86-AFH合成分子
<400> 341
ctgaagattc aagcttattt caataagact gcagacctgc catgccaatt tgcaaactct 60
caaaaccaaa gcctgagtga gctagtagta ttttggcagg accaggaaaa cttggttctg 120
aatgaggtat acttaggcaa agagaaattt gacagcgttc attccaagta tatgggccgc 180
acaagttttg attcagacag ttggaccctg agacttcaca atcttcagat caaggacaag 240
ggcttgtatc aatgtatcat ccatcacaaa aagcccacag gaatgattcg catccaccag 300
atgaattctg aactgtcagt gcttgctaac ttcagtcaac ctgaaatagt accaatttct 360
aatataacag aaaatgtgta cataaatttg acctgctcat ctatacacgg ttacccagaa 420
cctaagaaga tgagtgtttt gctaagaacc aagaattcaa ctatcgagta tgatggtatt 480
atgcagaaat ctcaagataa tgtcacagaa ctgtacgacg tttccatcag cttgtctgtt 540
tcattccctg atgttacgag caatatgacc atcttctgta ttctggaaac tgacaagacg 600
cggcttttat cttcaccttt ctctatagag cttgaggacc ctcagcctcc cccagactct 660
agactgaacg acatcttcga ggctcagaaa atcgaatggc acgaagatta caaggatgac 720
gacgataagg attacaagga tgacgacgat aaggattaca aggatgacga cgataagcat 780
catcatcatc atcactga 798
<210> 342
<211> 265
<212> PRT
<213> 人工序列
<220>
<223> 人CD86-AFH合成分子
<400> 342
Leu Lys Ile Gln Ala Tyr Phe Asn Lys Thr Ala Asp Leu Pro Cys Gln
1 5 10 15
Phe Ala Asn Ser Gln Asn Gln Ser Leu Ser Glu Leu Val Val Phe Trp
20 25 30
Gln Asp Gln Glu Asn Leu Val Leu Asn Glu Val Tyr Leu Gly Lys Glu
35 40 45
Lys Phe Asp Ser Val His Ser Lys Tyr Met Gly Arg Thr Ser Phe Asp
50 55 60
Ser Asp Ser Trp Thr Leu Arg Leu His Asn Leu Gln Ile Lys Asp Lys
65 70 75 80
Gly Leu Tyr Gln Cys Ile Ile His His Lys Lys Pro Thr Gly Met Ile
85 90 95
Arg Ile His Gln Met Asn Ser Glu Leu Ser Val Leu Ala Asn Phe Ser
100 105 110
Gln Pro Glu Ile Val Pro Ile Ser Asn Ile Thr Glu Asn Val Tyr Ile
115 120 125
Asn Leu Thr Cys Ser Ser Ile His Gly Tyr Pro Glu Pro Lys Lys Met
130 135 140
Ser Val Leu Leu Arg Thr Lys Asn Ser Thr Ile Glu Tyr Asp Gly Ile
145 150 155 160
Met Gln Lys Ser Gln Asp Asn Val Thr Glu Leu Tyr Asp Val Ser Ile
165 170 175
Ser Leu Ser Val Ser Phe Pro Asp Val Thr Ser Asn Met Thr Ile Phe
180 185 190
Cys Ile Leu Glu Thr Asp Lys Thr Arg Leu Leu Ser Ser Pro Phe Ser
195 200 205
Ile Glu Leu Glu Asp Pro Gln Pro Pro Pro Asp Ser Arg Leu Asn Asp
210 215 220
Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp Tyr Lys Asp Asp
225 230 235 240
Asp Asp Lys Asp Tyr Lys Asp Asp Asp Asp Lys Asp Tyr Lys Asp Asp
245 250 255
Asp Asp Lys His His His His His His
260 265
<210> 343
<211> 801
<212> DNA
<213> 人工序列
<220>
<223> 小鼠CD86-AFH合成分子
<400> 343
gtggagacgc aagcttattt caatgggact gcatatctgc cgtgcccatt tacaaaggct 60
caaaacataa gcctgagtga gctggtagta ttttggcagg accagcaaaa gttggttctg 120
tacgagcact atttgggcac agagaaactt gatagtgtga atgccaagta cctgggccgc 180
acgagctttg acaggaacaa ctggactcta cgacttcaca atgttcagat caaggacatg 240
ggctcgtatg attgttttat acaaaaaaag ccacccacag gatcaattat cctccaacag 300
acattaacag aactgtcagt gatcgccaac ttcagtgaac ctgaaataaa actggctcag 360
aatgtaacag gaaattctgg cataaatttg acctgcacgt ctaagcaagg tcacccgaaa 420
cctaagaaga tgtattttct gataactaat tcaactaatg agtatggtga taacatgcag 480
atatcacaag ataatgtcac agaactgttc agtatctcca acagcctctc tctttcattc 540
ccggatggtg tgtggcatat gaccgttgtg tgtgttctgg aaacggagtc aatgaagatt 600
tcctccaaac ctctcaattt cactcaagag tttccatctc ctcaaacgta ttggaaggag 660
tctagactga acgacatctt cgaggctcag aaaatcgaat ggcacgaaga ttacaaggat 720
gacgacgata aggattacaa ggatgacgac gataaggatt acaaggatga cgacgataag 780
catcatcatc atcatcactg a 801
<210> 344
<211> 266
<212> PRT
<213> 人工序列
<220>
<223> 小鼠CD86-AFH合成分子
<400> 344
Val Glu Thr Gln Ala Tyr Phe Asn Gly Thr Ala Tyr Leu Pro Cys Pro
1 5 10 15
Phe Thr Lys Ala Gln Asn Ile Ser Leu Ser Glu Leu Val Val Phe Trp
20 25 30
Gln Asp Gln Gln Lys Leu Val Leu Tyr Glu His Tyr Leu Gly Thr Glu
35 40 45
Lys Leu Asp Ser Val Asn Ala Lys Tyr Leu Gly Arg Thr Ser Phe Asp
50 55 60
Arg Asn Asn Trp Thr Leu Arg Leu His Asn Val Gln Ile Lys Asp Met
65 70 75 80
Gly Ser Tyr Asp Cys Phe Ile Gln Lys Lys Pro Pro Thr Gly Ser Ile
85 90 95
Ile Leu Gln Gln Thr Leu Thr Glu Leu Ser Val Ile Ala Asn Phe Ser
100 105 110
Glu Pro Glu Ile Lys Leu Ala Gln Asn Val Thr Gly Asn Ser Gly Ile
115 120 125
Asn Leu Thr Cys Thr Ser Lys Gln Gly His Pro Lys Pro Lys Lys Met
130 135 140
Tyr Phe Leu Ile Thr Asn Ser Thr Asn Glu Tyr Gly Asp Asn Met Gln
145 150 155 160
Ile Ser Gln Asp Asn Val Thr Glu Leu Phe Ser Ile Ser Asn Ser Leu
165 170 175
Ser Leu Ser Phe Pro Asp Gly Val Trp His Met Thr Val Val Cys Val
180 185 190
Leu Glu Thr Glu Ser Met Lys Ile Ser Ser Lys Pro Leu Asn Phe Thr
195 200 205
Gln Glu Phe Pro Ser Pro Gln Thr Tyr Trp Lys Glu Ser Arg Leu Asn
210 215 220
Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp Tyr Lys Asp
225 230 235 240
Asp Asp Asp Lys Asp Tyr Lys Asp Asp Asp Asp Lys Asp Tyr Lys Asp
245 250 255
Asp Asp Asp Lys His His His His His His
260 265
<210> 345
<211> 1116
<212> DNA
<213> 人工序列
<220>
<223> GITR Ig合成分子
<400> 345
cagcgcccca ccgggggtcc cgggtgcggc cctgggcgcc tcctgcttgg gacgggaacg 60
gacgcgcgct gctgccgggt tcacacgacg cgctgctgcc gcgattaccc gggcgaggag 120
tgctgttccg agtgggactg catgtgtgtc cagcctgaat tccactgcgg agacccttgc 180
tgcacgacct gccggcacca cccttgtccc ccaggccagg gggtacagtc ccaggggaaa 240
ttcagttttg gcttccagtg tatcgactgt gcctcgggga ccttctccgg gggccacgaa 300
ggccactgca aaccttggac agactgcacc cagttcgggt ttctcactgt gttccctggg 360
aacaagaccc acaacgctgt gtgcgtccca gggtccccgc cggcagagcc gcttgggctc 420
gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 480
ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 540
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 600
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 660
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 720
ggcaaggcgt acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 780
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 840
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 900
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 960
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1020
cggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1080
tacacgcaga agagcctctc cctgtctccg ggttaa 1116
<210> 346
<211> 371
<212> PRT
<213> 人工序列
<220>
<223> GITR Ig合成分子
<400> 346
Gln Arg Pro Thr Gly Gly Pro Gly Cys Gly Pro Gly Arg Leu Leu Leu
1 5 10 15
Gly Thr Gly Thr Asp Ala Arg Cys Cys Arg Val His Thr Thr Arg Cys
20 25 30
Cys Arg Asp Tyr Pro Gly Glu Glu Cys Cys Ser Glu Trp Asp Cys Met
35 40 45
Cys Val Gln Pro Glu Phe His Cys Gly Asp Pro Cys Cys Thr Thr Cys
50 55 60
Arg His His Pro Cys Pro Pro Gly Gln Gly Val Gln Ser Gln Gly Lys
65 70 75 80
Phe Ser Phe Gly Phe Gln Cys Ile Asp Cys Ala Ser Gly Thr Phe Ser
85 90 95
Gly Gly His Glu Gly His Cys Lys Pro Trp Thr Asp Cys Thr Gln Phe
100 105 110
Gly Phe Leu Thr Val Phe Pro Gly Asn Lys Thr His Asn Ala Val Cys
115 120 125
Val Pro Gly Ser Pro Pro Ala Glu Pro Leu Gly Leu Glu Pro Lys Ser
130 135 140
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
145 150 155 160
Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
165 170 175
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
180 185 190
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
195 200 205
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
210 215 220
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
225 230 235 240
Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro
245 250 255
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
260 265 270
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
275 280 285
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
290 295 300
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
305 310 315 320
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
325 330 335
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
340 345 350
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
355 360 365
Ser Pro Gly
370
<210> 347
<211> 1137
<212> DNA
<213> 人工序列
<220>
<223> PD1 Ig合成分子
<400> 347
ttagactccc cagacaggcc ctggaacccc cccaccttct ccccagccct gctcgtggtg 60
accgaagggg acaacgccac cttcacctgc agcttctcca acacatcgga gagcttcgtg 120
ctaaactggt accgcatgag ccccagcaac cagacggaca agctggccgc cttccccgag 180
gaccgcagcc agcccggcca ggactgccgc ttccgtgtca cacaactgcc caacgggcgt 240
gacttccaca tgagcgtggt cagggcccgg cgcaatgaca gcggcaccta cctctgtggg 300
gccatctccc tggcccccaa ggcgcagatc aaagagagcc tgcgggcaga gctcagggtg 360
acagagagaa gggcagaagt gcccacagcc caccccagcc cctcacccag gccagccggc 420
cagttccaac tcgagcagcc cagagggccc acaatcaagc cctgtcctcc atgcaaatgc 480
ccggctccaa atcttcttgg tggttcatcc gtcttcatct tccctccaaa gatcaaggat 540
gtactcatga tctccctgag ccccatagtc acatgtgtgg tggtggatgt gagcgaggac 600
gacccagatg tccagatcag ctggtttgtg aacaacgtgg aagtacacac agctcagaca 660
caaacccata gagaggatta caacagtact ctccgggtgg tcagtgccct ccccatccag 720
caccaggact ggatgagtgg caaggagttc aaatgcaagg tcaacaacaa agacctccca 780
gcgcccatcg agagaaccat ctcaaaaccc aaagggtcag taagagctcc acaggtatat 840
gtcttgcctc caccagaaga agagatgact aagaaacagg tcactctgac ctgcatggtc 900
acagacttca tgcctgaaga catttacgtg gagtggacta acaacgggaa aacagagcta 960
aactacaaga acactgaacc agtcctggac tctgatggtt cttacttcat gtacagcaag 1020
ctgagagtgg aaaagaagaa ctgggtggaa agaaatagct actcctgttc agtggtccac 1080
gagggtctgc acaatcacca cacgactaag agcttctccc ggactccggg taaataa 1137
<210> 348
<211> 378
<212> PRT
<213> 人工序列
<220>
<223> PD1 Ig合成分子
<400> 348
Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala
1 5 10 15
Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe
20 25 30
Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro
35 40 45
Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln
50 55 60
Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg
65 70 75 80
Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr
85 90 95
Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu
100 105 110
Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro
115 120 125
Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Leu
130 135 140
Glu Gln Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys
145 150 155 160
Pro Ala Pro Asn Leu Leu Gly Gly Ser Ser Val Phe Ile Phe Pro Pro
165 170 175
Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys
180 185 190
Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp
195 200 205
Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
210 215 220
Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln
225 230 235 240
His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn
245 250 255
Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly
260 265 270
Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu
275 280 285
Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met
290 295 300
Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu
305 310 315 320
Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe
325 330 335
Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
340 345 350
Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr
355 360 365
Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
370 375
<210> 349
<211> 1995
<212> DNA
<213> 人工序列
<220>
<223> 编码含H81接头的IL10 I87A::M0115融合蛋白的核苷酸序列
<400> 349
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacgc caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggtgaag ttcaaattcc cttgaccgaa 1200
agttacagcc cgaattctca ggtgcagctg gtgcagtctg gggctgaggt gaagaagcct 1260
ggggcctcag tgaaggtctc ctgcaaggct tctggataca ccttcaccga ctacaacatg 1320
aactgggtgc gacaggcccc tggacaaggg cttgagtgga tgggaaatat tgatccttac 1380
tatggtggta ctagttacaa tcagaagttc aagggcaggg tcaccatgac cagggacacg 1440
tccatcagca cagcctacat ggagctgagc aggctgagat ctgacgacac ggccgtgtat 1500
tactgtgcga gatgggacta taggtacgac gacgggaggg cttactatgt tatggacttc 1560
tggggccaag ggaccacggt caccgtctcc tcaggtggag gcggttcagg cggaggtgga 1620
tccggcggtg gcggatcggg tggcggcgga tctgacatcg tgatgaccca gtctccagac 1680
tccctggctg tgtctctggg cgagagggcc accatcaact gcaagtccag ccagagtgtt 1740
ttatacagct ccaaccagaa gaactactta gcttggtacc agcagaaacc aggacagcct 1800
cctaagctgc tcatttactg ggcatctacc cgggaatccg gggtccctga ccgattcagt 1860
ggcagcgggt ctgggacaga tttcactctc accatcagca gcctgcaggc tgaagatgtg 1920
gcagtttatt actgtcatca atacctctac tcgtggacgt ttggccaggg gaccaagctg 1980
gagatcaaac ggtaa 1995
<210> 350
<211> 664
<212> PRT
<213> 人工序列
<220>
<223> 含H81接头的IL10 I87A::M0115融合蛋白
<400> 350
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly Glu Val Gln Ile Pro Leu Thr Glu
385 390 395 400
Ser Tyr Ser Pro Asn Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu
405 410 415
Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
420 425 430
Tyr Thr Phe Thr Asp Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly
435 440 445
Gln Gly Leu Glu Trp Met Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr
450 455 460
Ser Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr
465 470 475 480
Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
485 490 495
Thr Ala Val Tyr Tyr Cys Ala Arg Trp Asp Tyr Arg Tyr Asp Asp Gly
500 505 510
Arg Ala Tyr Tyr Val Met Asp Phe Trp Gly Gln Gly Thr Thr Val Thr
515 520 525
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
530 535 540
Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp
545 550 555 560
Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser
565 570 575
Ser Gln Ser Val Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp
580 585 590
Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala
595 600 605
Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
610 615 620
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val
625 630 635 640
Ala Val Tyr Tyr Cys His Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gln
645 650 655
Gly Thr Lys Leu Glu Ile Lys Arg
660
<210> 351
<211> 1986
<212> DNA
<213> 人工序列
<220>
<223> 编码含H82接头的IL10 I87A::M0115融合蛋白的核苷酸序列
<400> 351
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacgc caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggtcacg acggctcttc cctgaataca 1200
ccgaattctc aggtgcagct ggtgcagtct ggggctgagg tgaagaagcc tggggcctca 1260
gtgaaggtct cctgcaaggc ttctggatac accttcaccg actacaacat gaactgggtg 1320
cgacaggccc ctggacaagg gcttgagtgg atgggaaata ttgatcctta ctatggtggt 1380
actagttaca atcagaagtt caagggcagg gtcaccatga ccagggacac gtccatcagc 1440
acagcctaca tggagctgag caggctgaga tctgacgaca cggccgtgta ttactgtgcg 1500
agatgggact ataggtacga cgacgggagg gcttactatg ttatggactt ctggggccaa 1560
gggaccacgg tcaccgtctc ctcaggtgga ggcggttcag gcggaggtgg atccggcggt 1620
ggcggatcgg gtggcggcgg atctgacatc gtgatgaccc agtctccaga ctccctggct 1680
gtgtctctgg gcgagagggc caccatcaac tgcaagtcca gccagagtgt tttatacagc 1740
tccaaccaga agaactactt agcttggtac cagcagaaac caggacagcc tcctaagctg 1800
ctcatttact gggcatctac ccgggaatcc ggggtccctg accgattcag tggcagcggg 1860
tctgggacag atttcactct caccatcagc agcctgcagg ctgaagatgt ggcagtttat 1920
tactgtcatc aatacctcta ctcgtggacg tttggccagg ggaccaagct ggagatcaaa 1980
cggtaa 1986
<210> 352
<211> 661
<212> PRT
<213> 人工序列
<220>
<223> 含H82接头的IL10 I87A::M0115融合蛋白
<400> 352
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly His Asp Gly Ser Ser Leu Asn Thr
385 390 395 400
Pro Asn Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
405 410 415
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
420 425 430
Thr Asp Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
435 440 445
Glu Trp Met Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn
450 455 460
Gln Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser
465 470 475 480
Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
485 490 495
Tyr Tyr Cys Ala Arg Trp Asp Tyr Arg Tyr Asp Asp Gly Arg Ala Tyr
500 505 510
Tyr Val Met Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala
545 550 555 560
Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser
565 570 575
Val Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln
580 585 590
Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
595 600 605
Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
610 615 620
Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr
625 630 635 640
Tyr Cys His Gln Tyr Leu Tyr Ser Trp Thr Phe Gly Gln Gly Thr Lys
645 650 655
Leu Glu Ile Lys Arg
660
<210> 353
<211> 2001
<212> DNA
<213> 人工序列
<220>
<223> 编码含H83接头的IL10 I87A::M0115融合蛋白的核苷酸序列
<400> 353
agcccaggcc agggcaccca gtctgagaac agctgcaccc acttcccagg caacctgcct 60
aacatgcttc gagatctccg agatgccttc agcagagtga agactttctt tcaaatgaag 120
gatcagctgg acaacttgtt gttaaaggag tccttgctgg aggactttaa gggttacctg 180
ggttgccaag ccttgtctga gatgatccag ttttacctgg aggaggtgat gccccaagct 240
gagaaccaag acccagacgc caaggcgcat gtgaactccc tgggggagaa cctgaagacc 300
ctcaggctga ggctacggcg ctgtcatcga tttcttccct gtgaaaacaa gagcaaggcc 360
gtggagcagg tgaagaatgc ctttaataag ctccaagaga aaggcatcta caaagccatg 420
agtgagtttg acatcttcat caactacata gaagcctaca ttacaatgaa gatacgaaac 480
ctcgagccca aatcttctga caaaactcac acatgcccac cgtgcccagc acctgaagcc 540
gcgggtgcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 600
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 660
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 720
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 780
aatggcaagg cgtacgcgtg cgcggtctcc aacaaagccc tcccagcccc catcgagaaa 840
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 900
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatcca 960
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1020
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1080
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1140
cactacacgc agaagagcct ctccctgtct ccgggttctt ccctgaatac aggaactcag 1200
atggcaggtc attctccgaa ttctcaggtg cagctggtgc agtctggggc tgaggtgaag 1260
aagcctgggg cctcagtgaa ggtctcctgc aaggcttctg gatacacctt caccgactac 1320
aacatgaact gggtgcgaca ggcccctgga caagggcttg agtggatggg aaatattgat 1380
ccttactatg gtggtactag ttacaatcag aagttcaagg gcagggtcac catgaccagg 1440
gacacgtcca tcagcacagc ctacatggag ctgagcaggc tgagatctga cgacacggcc 1500
gtgtattact gtgcgagatg ggactatagg tacgacgacg ggagggctta ctatgttatg 1560
gacttctggg gccaagggac cacggtcacc gtctcctcag gtggaggcgg ttcaggcgga 1620
ggtggatccg gcggtggcgg atcgggtggc ggcggatctg acatcgtgat gacccagtct 1680
ccagactccc tggctgtgtc tctgggcgag agggccacca tcaactgcaa gtccagccag 1740
agtgttttat acagctccaa ccagaagaac tacttagctt ggtaccagca gaaaccagga 1800
cagcctccta agctgctcat ttactgggca tctacccggg aatccggggt ccctgaccga 1860
ttcagtggca gcgggtctgg gacagatttc actctcacca tcagcagcct gcaggctgaa 1920
gatgtggcag tttattactg tcatcaatac ctctactcgt ggacgtttgg ccaggggacc 1980
aagctggaga tcaaacggta a 2001
<210> 354
<211> 666
<212> PRT
<213> 人工序列
<220>
<223> 含H83接头的IL10 I87A::M0115融合蛋白
<400> 354
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Ile Thr Met Lys Ile Arg Asn
145 150 155 160
Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly Ser Ser Leu Asn Thr Gly Thr Gln
385 390 395 400
Met Ala Gly His Ser Pro Asn Ser Gln Val Gln Leu Val Gln Ser Gly
405 410 415
Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
420 425 430
Ser Gly Tyr Thr Phe Thr Asp Tyr Asn Met Asn Trp Val Arg Gln Ala
435 440 445
Pro Gly Gln Gly Leu Glu Trp Met Gly Asn Ile Asp Pro Tyr Tyr Gly
450 455 460
Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Met Thr Arg
465 470 475 480
Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser
485 490 495
Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Asp Tyr Arg Tyr Asp
500 505 510
Asp Gly Arg Ala Tyr Tyr Val Met Asp Phe Trp Gly Gln Gly Thr Thr
515 520 525
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser
545 550 555 560
Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys
565 570 575
Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu
580 585 590
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr
595 600 605
Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
610 615 620
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
625 630 635 640
Asp Val Ala Val Tyr Tyr Cys His Gln Tyr Leu Tyr Ser Trp Thr Phe
645 650 655
Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
660 665
<210> 355
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> H68接头
<400> 355
Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr
1 5 10 15
Ser Pro Asn Ser
20
<210> 356
<211> 26
<212> PRT
<213> 人工序列
<220>
<223> H69接头
<400> 356
Glu Gln Lys Leu Ser Asn Met Glu Asn Arg Leu Lys Pro Phe Phe Thr
1 5 10 15
Ser Gly Ser Ala Asp Thr Ser Pro Asn Ser
20 25
<210> 357
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> H77接头
<400> 357
Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Pro Asn
1 5 10 15
Ser
<210> 358
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> H78接头
<400> 358
Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Pro Asn Ser
1 5 10
<210> 359
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> H79接头
<400> 359
Asn Ser Leu Phe Asn Gln Glu Val Pro Asn Ser
1 5 10
<210> 360
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> H80接头
<400> 360
Gln Arg His Asn Asn Ser Ser Leu Asn Thr Pro Asn Ser
1 5 10
<210> 361
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> H81接头
<400> 361
Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser
1 5 10
<210> 362
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> H82接头
<400> 362
His Asp Gly Ser Ser Leu Asn Thr Pro Asn Ser
1 5 10
<210> 363
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> H83接头
<400> 363
Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Gly His Ser Pro Asn Ser
1 5 10 15
<210> 364
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> A2铰链
<400> 364
Pro Pro Pro Pro Pro Cys Pro Pro Cys
1 5
<210> 365
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> H75接头
<400> 365
Gln Arg His Asn Asn Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Gly
1 5 10 15
His Ser Pro Asn Ser
20
<210> 366
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> H75a接头
<400> 366
Gln Arg His Asn Asn Ser Ser Leu Asn Thr Gly Thr Gln Lys Ala Arg
1 5 10 15
His Ser Pro Asn Ser
20
<210> 367
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> H75b接头
<400> 367
Gln Arg His Asn Asn Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Arg
1 5 10 15
His Ser Pro Asn Ser
20
<210> 368
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> H75c接头
<400> 368
Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gln Lys Ala Gly
1 5 10 15
His Ser Pro Asn Ser
20
<210> 369
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> H75d接头
<400> 369
Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gln Met Ala Gly
1 5 10 15
His Ser Pro Asn Ser
20
<210> 370
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> H75e接头
<400> 370
Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gln Met Ala Arg
1 5 10 15
His Ser Pro Asn Ser
20
<210> 371
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> H75f接头
<400> 371
Gln Arg His Ala Asn Ser Ala Leu Asn Thr Gly Thr Gln Met Ala Gly
1 5 10 15
His Ser Pro Asn Ser
20
<210> 372
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> H75g接头
<400> 372
Gln Arg His Asn Ala Ala Ser Leu Asn Thr Gly Thr Gln Met Ala Gly
1 5 10 15
His Ser Pro Asn Ser
20
<210> 373
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> H75h接头
<400> 373
Gln Arg His Ala Ala Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Gly
1 5 10 15
His Ser Pro Asn Ser
20
<210> 374
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> H68a接头
<400> 374
Asn Ser Leu Ala Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr
1 5 10 15
Ser Pro Asn Ser
20
<210> 375
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> H68b接头
<400> 375
Asn Ser Gly Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr
1 5 10 15
Ser Pro Asn Ser
20
<210> 376
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> H68c接头
<400> 376
Asn Ser Gly Ala Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr
1 5 10 15
Ser Pro Asn Ser
20
<210> 377
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> H68s接头
<400> 377
Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser
1 5 10 15
<210> 378
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 可变区接头亚基
<400> 378
Gly Gly Gly Gly Ser
1 5
<210> 379
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 单IL10接头
<400> 379
Gly Gly Gly Ser Gly Gly
1 5
<210> 380
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 单IL10合成分子
<400> 380
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu
115 120 125
Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
130 135 140
Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met
145 150 155 160
Thr Met Lys Ile Arg Asn
165
<210> 381
<211> 160
<212> PRT
<213> 人工序列
<220>
<223> IL10 I87A合成分子
<400> 381
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ala Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
<210> 382
<211> 160
<212> PRT
<213> 人工序列
<220>
<223> IL10 I87S合成分子
<400> 382
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ser Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
<210> 383
<211> 10
<212> PRT
<213> 智人(Homo sapiens)
<400> 383
Glu Pro Lys Ser Cys Asp Lys Thr His Thr
1 5 10
<210> 384
<211> 7
<212> PRT
<213> 智人(Homo sapiens)
<400> 384
Glu Arg Lys Cys Cys Val Glu
1 5
<210> 385
<211> 12
<212> PRT
<213> 智人(Homo sapiens)
<400> 385
Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr
1 5 10
<210> 386
<211> 10
<212> PRT
<213> 智人(Homo sapiens)
<400> 386
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro
1 5 10
<210> 387
<211> 7
<212> PRT
<213> 智人(Homo sapiens)
<400> 387
Glu Ser Lys Tyr Gly Pro Pro
1 5
<210> 388
<211> 5
<212> PRT
<213> 智人(Homo sapiens)
<400> 388
Cys Pro Arg Cys Pro
1 5
<210> 389
<211> 5
<212> PRT
<213> 智人(Homo sapiens)
<400> 389
Cys Pro Ser Cys Pro
1 5
<210> 390
<211> 17
<212> PRT
<213> 智人(Homo sapiens)
<400> 390
Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys
1 5 10 15
Pro
<210> 391
<211> 15
<212> PRT
<213> 智人(Homo sapiens)
<400> 391
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
1 5 10 15
<210> 392
<211> 34
<212> PRT
<213> 智人(Homo sapiens)
<400> 392
Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln
1 5 10 15
Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg
20 25 30
Asn Thr
<210> 393
<211> 24
<212> PRT
<213> 智人(Homo sapiens)
<400> 393
Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln
1 5 10 15
Glu Glu Arg Glu Thr Lys Thr Pro
20
<210> 394
<211> 19
<212> PRT
<213> 智人(Homo sapiens)
<400> 394
Val Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro
1 5 10 15
Ser Pro Ser
<210> 395
<211> 6
<212> PRT
<213> 智人(Homo sapiens)
<400> 395
Val Pro Pro Pro Pro Pro
1 5
<210> 396
<211> 107
<212> PRT
<213> 智人(Homo sapiens)
<400> 396
Val Cys Ser Arg Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser
1 5 10 15
Ser Ser Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys
20 25 30
Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu
35 40 45
Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln
50 55 60
Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys
65 70 75 80
His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly
85 90 95
His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala
100 105
<210> 397
<211> 112
<212> PRT
<213> 智人(Homo sapiens)
<400> 397
Val Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg
1 5 10 15
Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala
20 25 30
Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly
35 40 45
Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala
50 55 60
Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile
65 70 75 80
Lys Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr Cys Arg Val Asp
85 90 95
His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro
100 105 110
<210> 398
<211> 5
<212> PRT
<213> 智人(Homo sapiens)
<400> 398
Cys Pro Pro Cys Pro
1 5
<210> 399
<211> 1614
<212> DNA
<213> 人工序列
<220>
<223> 编码CTLA4 x IL10 IgA融合蛋白的核苷酸序列
<400> 399
atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg 60
tgtgagtatg catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct 120
gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc 180
ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa 240
ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg 300
ccatactacc tgggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc 360
ccagattctg acctcgagcc caaatcttct gacaaaactc acacatgccc accgtgccca 420
gcacctgaag ccgcgggtgc accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 480
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 540
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 600
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660
caggactggc tgaatggcaa ggcgtacgcg tgcgcggtct ccaacaaagc cctcccagcc 720
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 780
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 840
ggcttctatc caagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 900
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 960
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1020
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtcc ctcaactcca 1080
cctaccccat ctccctcaac tccacctacc ccatctccct caccgaattc tagcccaggc 1140
cagggcaccc agtctgagaa cagctgcacc cacttcccag gcaacctgcc taacatgctt 1200
cgagatctcc gagatgcctt cagcagagtg aagactttct ttcaaatgaa ggatcagctg 1260
gacaacttgt tgttaaagga gtccttgctg gaggacttta agggttacct gggttgccaa 1320
gccttgtctg agatgatcca gttttacctg gaggaggtga tgccccaagc tgagaaccaa 1380
gacccagaca tcaaggcgca tgtgaactcc ctgggggaga acctgaagac cctcaggctg 1440
aggctacggc gctgtcatcg atttcttccc tgtgaaaaca agagcaaggc cgtggagcag 1500
gtgaagaatg cctttaataa gctccaagag aaaggcatct acaaagccat gagtgagttt 1560
gacatcttca tcaactacat agaagcctac atgacaatga agatacgaaa ctaa 1614
<210> 400
<211> 537
<212> PRT
<213> 人工序列
<220>
<223> CTLA4 x IL10 IgA融合蛋白
<400> 400
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
130 135 140
Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro
355 360 365
Pro Thr Pro Ser Pro Ser Pro Asn Ser Ser Pro Gly Gln Gly Thr Gln
370 375 380
Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu
385 390 395 400
Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met
405 410 415
Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp
420 425 430
Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe
435 440 445
Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile
450 455 460
Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu
465 470 475 480
Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys
485 490 495
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly
500 505 510
Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu
515 520 525
Ala Tyr Met Thr Met Lys Ile Arg Asn
530 535
<210> 401
<211> 1530
<212> DNA
<213> 人工序列
<220>
<223> M0115 PIMS合成分子
<400> 401
gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 60
ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggcgt acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaactcat tattcaacca agaagttcaa 720
attcccttga ccgaaagtta cagcccgaat tctcaggtgc agctggtgca gtctggggct 780
gaggtgaaga agcctggggc ctcagtgaag gtctcctgca aggcttctgg atacaccttc 840
accgactaca acatgaactg ggtgcgacag gcccctggac aagggcttga gtggatggga 900
aatattgatc cttactatgg tggtactagt tacaatcaga agttcaaggg cagggtcacc 960
atgaccaggg acacgtccat cagcacagcc tacatggagc tgagcaggct gagatctgac 1020
gacacggccg tgtattactg tgcgagatgg gactataggt acgacgacgg gagggcttac 1080
tatgttatgg acttctgggg ccaagggacc acggtcaccg tctcctcagg tggaggcggt 1140
tcaggcggag gtggatccgg cggtggcgga tcgggtggcg gcggatctga catcgtgatg 1200
acccagtctc cagactccct ggctgtgtct ctgggcgaga gggccaccat caactgcaag 1260
tccagccaga gtgttttata cagctccaac cagaagaact acttagcttg gtaccagcag 1320
aaaccaggac agcctcctaa gctgctcatt tactgggcat ctacccggga atccggggtc 1380
cctgaccgat tcagtggcag cgggtctggg acagatttca ctctcaccat cagcagcctg 1440
caggctgaag atgtggcagt ttattactgt catcaatacc tctactcgtg gacgtttggc 1500
caggggacca agctggagat caaacggtaa 1530
<210> 402
<211> 509
<212> PRT
<213> 人工序列
<220>
<223> M0115 PIMS合成分子
<400> 402
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln
225 230 235 240
Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Gln Val Gln Leu Val
245 250 255
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser
260 265 270
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Asn Met Asn Trp Val
275 280 285
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Asn Ile Asp Pro
290 295 300
Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr
305 310 315 320
Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg
325 330 335
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Asp Tyr
340 345 350
Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met Asp Phe Trp Gly Gln
355 360 365
Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
370 375 380
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met
385 390 395 400
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr
405 410 415
Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Gln Lys
420 425 430
Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu
435 440 445
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe
450 455 460
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
465 470 475 480
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln Tyr Leu Tyr Ser
485 490 495
Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
500 505
<210> 403
<211> 1095
<212> DNA
<213> 人工序列
<220>
<223> 编码小鼠CTLA4与人Fc结构域的融合物的核苷酸序列
<400> 403
gccatacagg tgacccaacc ttcagtggtg ttggctagca gccatggtgt cgccagcttt 60
ccatgtgaat attcaccatc acacaacact gatgaggtcc gggtgactgt gctgcggcag 120
acaaatgacc aaatgactga ggtctgtgcc acgacattca cagagaagaa tacagtgggc 180
ttcctagatt accccttctg cagtggtacc tttaatgaaa gcagagtgaa cctcaccatc 240
caaggactga gagctgttga cacgggactg tacctctgca aggtggaact catgtaccca 300
ccgccatact ttgtgggcat gggcaacggg acgcagattt atgtcattga tccagaacca 360
tgcccggatt ctgacctcga gcccagagtg cccataacac agaacccctg tcctccactc 420
aaagagtgtc ccccatgcgc agctccagac gcagcgggtg cgccatccgt cttcatcttc 480
cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 540
gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 600
gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 660
agtgccctcc ccatccagca ccaggactgg atgagtggca aggcgttcgc atgcgcggtc 720
aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 780
agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 840
agtctgacct gcatgatcgc aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 900
aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 960
tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1020
gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1080
tctctgggta aataa 1095
<210> 404
<211> 364
<212> PRT
<213> 人工序列
<220>
<223> 小鼠CTLA4与人Fc结构域的融合物
<400> 404
Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser His Gly
1 5 10 15
Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His Asn Thr Asp Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln Met Thr Glu Val
35 40 45
Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp Tyr
50 55 60
Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Leu Glu Pro
115 120 125
Arg Val Pro Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu Cys Pro
130 135 140
Pro Cys Ala Ala Pro Asp Ala Ala Gly Ala Pro Ser Val Phe Ile Phe
145 150 155 160
Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Met Val
165 170 175
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile
180 185 190
Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
195 200 205
His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro
210 215 220
Ile Gln His Gln Asp Trp Met Ser Gly Lys Ala Phe Ala Cys Ala Val
225 230 235 240
Asn Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro
245 250 255
Arg Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Ala
260 265 270
Glu Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys Met Ile Ala Gly
275 280 285
Phe Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn Gly Arg Thr
290 295 300
Glu Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser Asp Gly Ser
305 310 315 320
Tyr Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr Trp Glu Arg
325 330 335
Gly Ser Leu Phe Ala Cys Ser Val Val His Glu Gly Leu His Asn His
340 345 350
Leu Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly Lys
355 360
<210> 405
<211> 570
<212> DNA
<213> 人工序列
<220>
<223> 编码与C末端AviTag融合的PD1胞外域的核苷酸序列
<400> 405
ttagactccc cagacaggcc ctggaacccc cccaccttct ccccagccct gctcgtggtg 60
accgaagggg acaacgccac cttcacctgc agcttctcca acacatcgga gagcttcgtg 120
ctaaactggt accgcatgag ccccagcaac cagacggaca agctggccgc cttccccgag 180
gaccgcagcc agcccggcca ggactgccgc ttccgtgtca cacaactgcc caacgggcgt 240
gacttccaca tgagcgtggt cagggcccgg cgcaatgaca gcggcaccta cctctgtggg 300
gccatctccc tggcccccaa ggcgcagatc aaagagagcc tgcgggcaga gctcagggtg 360
acagagagaa gggcagaagt gcccacagcc caccccagcc cctcacccag gccagccggc 420
cagttccaat ctagactgaa cgacatcttc gaggctcaga aaatcgaatg gcacgaagat 480
tacaaggatg acgacgataa ggattacaag gatgacgacg ataaggatta caaggatgac 540
gacgataagc atcatcatca tcatcactga 570
<210> 406
<211> 189
<212> PRT
<213> 人工序列
<220>
<223> 与C末端AviTag融合的PD1胞外域
<400> 406
Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala
1 5 10 15
Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe
20 25 30
Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro
35 40 45
Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln
50 55 60
Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg
65 70 75 80
Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr
85 90 95
Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu
100 105 110
Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro
115 120 125
Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Ser
130 135 140
Arg Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp
145 150 155 160
Tyr Lys Asp Asp Asp Asp Lys Asp Tyr Lys Asp Asp Asp Asp Lys Asp
165 170 175
Tyr Lys Asp Asp Asp Asp Lys His His His His His His
180 185
<210> 407
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> H75i接头
<400> 407
Gln Arg His Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Gly His Ser
1 5 10 15
Pro Asn Ser
<210> 408
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> H75j接头
<400> 408
Gln Arg His Gly Gly Ser Ser Leu Asn Thr Gly Thr Gln Met Ala Gly
1 5 10 15
His Ser Pro Asn Ser
20
<210> 409
<211> 217
<212> PRT
<213> 人工序列
<220>
<223> 人IgG1 CH2和CH3区
<400> 409
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
115 120 125
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 410
<211> 124
<212> PRT
<213> 智人(Homo sapiens)
<400> 410
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp
115 120
<210> 411
<211> 124
<212> PRT
<213> 人工序列
<220>
<223> 含A29Y L104E突变的CTLA4
<400> 411
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp
115 120
<210> 412
<211> 134
<212> PRT
<213> 智人(Homo sapiens)
<400> 412
Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp Asn
1 5 10 15
Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg Glu
20 25 30
Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu Val Cys
35 40 45
Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys Thr
50 55 60
Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr
65 70 75 80
Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile
85 90 95
Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly
100 105 110
Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe
115 120 125
Pro Gly Pro Ser Lys Pro
130
<210> 413
<211> 1530
<212> DNA
<213> 人工序列
<220>
<223> FUN1 PIMS M0115合成分子
<400> 413
gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgcg 60
ggtgcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggcgt acgcgtgcgc ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatccaagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaactcat tattcaacca agaagttcaa 720
attcccttga ccgaaagtta cagcccgaat tctcaggtgc agctggtgca gtctggggct 780
gaggtgaaga agcctggggc ctcagtgaag gtctcctgca aggcttctgg atacaccttc 840
accgactaca acatgaactg ggtgcgacag gcccctggac aagggcttga gtggatggga 900
aatattgatc cttactatgg tggtactagt tacaatcaga agttcaaggg cagggtcacc 960
atgaccaggg acacgtccat cagcacagcc tacatggagc tgagcaggct gagatctgac 1020
gacacggccg tgtattactg tgcgagatgg gactataggt acgacgacgg gagggcttac 1080
tatgttatgg acttctgggg ccaagggacc acggtcaccg tctcctcagg tggaggcggt 1140
tcaggcggag gtggatccgg cggtggcgga tcgggtggcg gcggatctga catcgtgatg 1200
acccagtctc cagactccct ggctgtgtct ctgggcgaga gggccaccat caactgcaag 1260
tccagccaga gtgttttata cagctccaac cagaagaact acttagcttg gtaccagcag 1320
aaaccaggac agcctcctaa gctgctcatt tactgggcat ctacccggga atccggggtc 1380
cctgaccgat tcagtggcag cgggtctggg acagatttca ctctcaccat cagcagcctg 1440
caggctgaag atgtggcagt ttattactgt catcaatacc tctactcgtg gacgtttggc 1500
caggggacca agctggagat caaacggtaa 1530
<210> 414
<211> 509
<212> PRT
<213> 人工序列
<220>
<223> FUN1 PIMS M0115合成分子
<400> 414
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Asn Ser Leu Phe Asn Gln Glu Val Gln
225 230 235 240
Ile Pro Leu Thr Glu Ser Tyr Ser Pro Asn Ser Gln Val Gln Leu Val
245 250 255
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser
260 265 270
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Asn Met Asn Trp Val
275 280 285
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Asn Ile Asp Pro
290 295 300
Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr
305 310 315 320
Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg
325 330 335
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Asp Tyr
340 345 350
Arg Tyr Asp Asp Gly Arg Ala Tyr Tyr Val Met Asp Phe Trp Gly Gln
355 360 365
Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
370 375 380
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met
385 390 395 400
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr
405 410 415
Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Gln Lys
420 425 430
Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu
435 440 445
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe
450 455 460
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
465 470 475 480
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln Tyr Leu Tyr Ser
485 490 495
Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
500 505
<210> 415
<211> 216
<212> PRT
<213> 人工序列
<220>
<223> 人IgG1 CH2CH3 脱Lys(desLys)
<400> 415
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
115 120 125
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly
210 215
<210> 416
<211> 217
<212> PRT
<213> 人工序列
<220>
<223> 人IgG1 CH2CH3无效2
<400> 416
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
115 120 125
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 417
<211> 216
<212> PRT
<213> 人IgG1 CH2CH3无效2 脱Lys人工序列
<220>
<223> 人IgG1 CH2CH3无效2 脱Lys
<400> 417
Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
115 120 125
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly
210 215
<210> 418
<211> 160
<212> PRT
<213> 智人(Homo sapiens)
<400> 418
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
<210> 419
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> 异源前导序列
<220>
<221> 变体
<222> 23
<223> Xaa = 任何氨基酸
<400> 419
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Xaa
20
<210> 420
<211> 24
<212> PRT
<213> 人工序列
<220>
<223> 异源前导序列
<220>
<221> 变体
<222> 23, 24
<223> Xaa = 任何氨基酸
<400> 420
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Xaa Xaa
20
<210> 421
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 异源前导序列
<220>
<221> 变体
<222> 23, 24, 25
<223> Xaa = 任何氨基酸
<400> 421
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Xaa Xaa Xaa
20 25
<210> 422
<211> 4
<212> PRT
<213> 未知
<220>
<223> 产生形成二硫键能力的天然产生或添加的基序
<400> 422
Cys Pro Pro Cys
1

Claims (35)

1.一种多特异性融合蛋白，包含通过间插结构域与异源结合域连接的CD86结合域，其中所述异源结合域是IL-10激动剂、HLA-G激动剂、HGF激动剂、IL-35激动剂、PD-1激动剂、BTLA激动剂、LIGHT拮抗物、GITRL拮抗物或CD40拮抗物。
2.如权利要求1所述的多特异性融合蛋白，其特征在于，所述CD86结合域是CTLA4胞外域或CTLA4胞外域的亚结构域。
3.如权利要求1所述的多特异性融合蛋白，其特征在于，所述CD86结合域是具有CD86特异性的Fab、scFv、结构域抗体或仅含重链的抗体。
4.如权利要求3所述的多特异性融合蛋白，其特征在于，所述CD86结合域包含FUN1抗CD86抗体或其人源化变体的轻链和重链可变结构域。
5.如权利要求4所述的多特异性融合蛋白，其特征在于，所述FUN1结合域包含SEQ IDNO:187或237的氨基酸1-258。
6.如权利要求1所述的多特异性融合蛋白，其特征在于，所述CD86结合域包含SEQ IDNO:1-6中任一项所列的氨基酸序列。
7.如权利要求1所述的多特异性融合蛋白，其特征在于，所述异源结合域包含SEQ IDNO:7、14、15、18-22、25、26、29、32、33、39和40中任一项所列的氨基酸序列。
8.如权利要求1所述的多特异性融合蛋白，其特征在于，所述异源结合域包含IL10激动剂，所述IL10激动剂包含SEQ ID NO:7或其包含第87位点突变的变体提供的氨基酸序列。
9.如权利要求1所述的多特异性融合蛋白，其特征在于，所述异源结合域包含HLA-G激动剂，所述HLA-G激动剂包含SEQ ID NO:14或15提供的氨基酸序列。
10.如权利要求1所述的多特异性融合蛋白，其特征在于，所述异源结合域包含HGF激动剂，所述HGF激动剂包含SEQ ID NO:18-22中任一项提供的氨基酸序列。
11.如权利要求1所述的多特异性融合蛋白，其特征在于，所述异源结合域包含IL-35激动剂，所述IL-35激动剂包含SEQ ID NO:25或26提供的氨基酸序列。
12.如权利要求1所述的多特异性融合蛋白，其特征在于，所述异源结合域包含PD-1激动剂，所述PD-1激动剂包含SEQ ID NO:32或33提供的氨基酸序列。
13.如权利要求1所述的多特异性融合蛋白，其特征在于，所述异源结合域包含BTLA激动剂，所述BTLA激动剂包含SEQ ID NO:29提供的氨基酸序列。
14.如权利要求1所述的多特异性融合蛋白，其特征在于，所述异源结合域包含LIGHT拮抗物，所述LIGHT拮抗物包含SEQ ID NO:29提供的氨基酸序列。
15.如权利要求1所述的多特异性融合蛋白，其特征在于，所述异源结合域包含GITRL拮抗物，所述GITRL拮抗物包含SEQ ID NO:39或40提供的氨基酸序列。
16.如权利要求1所述的多特异性融合蛋白，其特征在于，所述间插结构域包含置于CD86结合域和异源结合域之间的免疫球蛋白恒定区或亚区。
17.如权利要求16所述的多特异性融合蛋白，其特征在于，所述免疫球蛋白恒定区或亚区是IgG1CH2CH3。
18.如权利要求1所述的多特异性融合蛋白，其特征在于，所述间插结构域包含置于第一和第二接头之间的免疫球蛋白恒定区。
19.如权利要求18所述的多特异性融合蛋白，其特征在于，所述第一和第二接头独立地选自SEQ ID NO:43-166、244、307、320、355-379和383-398提供的接头。
20.如权利要求18所述的多特异性融合蛋白，其特征在于，所述间插结构域包含人免疫球蛋白Fc区、清蛋白、转铁蛋白或结合血清蛋白的支架结构域。
21.如权利要求1所述的多特异性融合蛋白，其特征在于，所述间插结构域从氨基末端到羧基末端包含如下结构：
-L1-X-L2-
其中：
L1和L2各自独立为包含2到约150个氨基酸的接头；和
X是免疫球蛋白恒定区或亚区、清蛋白、转铁蛋白或另一血清蛋白结合蛋白。
22.如权利要求21所述的多特异性融合蛋白，其特征在于，所述免疫球蛋白恒定区或亚区是IgG1CH2CH3。
23.如权利要求21所述的多特异性融合蛋白，其特征在于，L1是人免疫球蛋白铰链区，其经任选突变用其它氨基酸取代一个或多个半胱氨酸。
24.如权利要求21或23所述的多特异性融合蛋白，其特征在于，X是人IgG1Fc结构域或它的至少一个CH结构域，其经任选突变以消除FcγRI-III相互作用并保持FcRn相互作用。
25.如权利要求1所述的多特异性融合蛋白，其特征在于，所述间插结构域是二聚化结构域。
26.如权利要求1所述的多特异性融合蛋白，其特征在于，其具有以下结构：
N-BD1-X-L2-BD2-C
其中：
BD1是与CTLA4胞外域至少约90％相同的CD86结合域；
-X-是-L1-CH2CH3-，其中L1是第一IgG1铰链，其任选通过取代第一半胱氨酸而发生突变，且-CH2CH3-是IgG1Fc结构域的CH2CH3区，其经任选突变以消除FcγRI-III相互作用并保持FcRn相互作用；
L2是选自SEQ ID NO:43-166、244、307、320、355-379和383-398的接头；和
BD2是异源结合域。
27.如权利要求1所述的多特异性融合蛋白，其特征在于，所述融合蛋白包含以下任一项提供的氨基酸序列：SEQ ID NO:9、13、17、24、28、31、35、38、42、171、173、175、177、179、181、183、185、187、189、191、193、195、197、199、201、203、205、207、209、211、213、215、217、219、221、223、237、239、252、254、256、258、260、262、266、276、302、330、334、336、338、340、350、352和354。
28.一种组合物，其包含以上权利要求中任一项所述的一种或多种多特异性融合蛋白，以及药学上可接受的载体、稀释剂或赋形剂。
29.如权利要求28所述的组合物，其特征在于，所述多特异性融合蛋白在所述组合物中以二聚体或多聚体形式存在。
30.一种多核苷酸，其编码如权利要求1-27中任一项所述的多特异性融合蛋白。
31.如权利要求30所述的多核苷酸，其特征在于，所述多核苷酸包含以下任一项提供的多核苷酸：SEQ ID NO:8、12、16、23、27、30、34、37、41、170、172、174、176、178、180、182、184、186、188、190、192、194、196、198、200、202、204、206、208、210、212、214、216、218、220、222、236、238、251、253、255、257、259、261、265、275、301、329、333、335、337、339、349、351和353。
32.一种表达载体，其包含与表达控制序列操作性连接的如权利要求30或31所述的多核苷酸。
33.一种宿主细胞，其包含权利要求32所述的表达载体。
34.一种治疗患与CD86、IL-10、HLA-G、HGF、IL-35、PD-1、BTLA、LIGHT、GITRL或CD40相关疾病的对象的方法，包括给予治疗有效量的以上权利要求中任一项所述的多特异性融合蛋白或其组合物。
35.如权利要求34所述的方法，其特征在于，所述疾病是类风湿性关节炎、青少年类风湿性关节炎、系统性红斑狼疮或实体器官移植。
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