CN112074532A - 结合非天然NKG2D受体的非天然NKG2D配体的修饰的α1-α2结构域 - Google Patents

结合非天然NKG2D受体的非天然NKG2D配体的修饰的α1-α2结构域 Download PDF

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CN112074532A
CN112074532A CN201980022711.XA CN201980022711A CN112074532A CN 112074532 A CN112074532 A CN 112074532A CN 201980022711 A CN201980022711 A CN 201980022711A CN 112074532 A CN112074532 A CN 112074532A
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卡曼·C·金姆
凯尔·E·兰德格拉夫
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Xyphos Biosciences Inc
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Abstract

本公开总体涉及与非天然NKG2D受体的非天然胞外域特异性结合的，NKG2D配体的修饰的α1‑α2结构域所组成的多肽的制备，并且其中异源分子连接到NKG2D配体的修饰的1‑2个结构域。本公开还涉及与包括多肽，和在一些实施例中抗体或抗体片段，的异源分子连接的NKG2D配体的修饰的α1‑α2结构域。本公开还涉及NKG2D受体的修饰形式，其被工程化以分别提供与非天然和天然形式的NKG2D配体的增强和减弱结合的组合。

Description

结合非天然NKG2D受体的非天然NKG2D配体的修饰的α1-α2结 构域
技术领域
本申请总体涉及与非天然NKG2D受体的非天然胞外域特异性结合的，NKG2D配体的修饰的α1-α2结构域所组成的多肽的制备，并且其中异源分子连接到NKG2D配体的修饰的α1-α2结构域。
背景技术
NKG2D是在自然杀伤(NK)细胞和某些T细胞表面上表达为II型同源二聚体整合蛋白的活化受体。当NKG2D与主要表达在受损细胞表面的8种天然配体中的一种结合时，NKG2D激活的NK细胞杀死应激细胞，或者当NKG2D与T细胞结合时，配体占据的NKG2D共同刺激T细胞执行其效应子功能。已经解决了人类天然NKG2D的胞外域其可溶性天然配体中的几种以及在一些情况下可溶性配体和受体胞外域的结合复合物，的三维结构。NKG2D配体的单体α1-α2结构域与天然NKG2D同源二聚体的两个胞外域特异性结合。
发明内容
本公开涉及与包括多肽，在一些实施例中抗体或抗体片段，的异源分子连接的NKG2D配体的修饰的α1-α2结构域。本发明还涉及NKG2D受体的修饰形式，其被工程化以分别提供与非天然和天然形式的NKG2D配体的增强和减弱的结合的组合。
附图说明
图1A：天然NKG2D.wt胞外域(SEQ ID NO:17)与NKG2D.YA(SEQ ID NO:18)和NKG2D.AF(SEQ ID NO:25)非天然变体的比对。显示的是Y152和Y199的位置，灰色突出显示的是非天然变体中存在的突变残基。
图1B：天然/野生型ULBP2的α1-α2结构域(SEQ ID NO:4)和ULBP2(包括ULBP2.R80W)的非天然变体(SEQ ID NO:108)的比对。灰色突出显示的是非天然ULBP2变体与非天然NKG2D.YA或NKG2D.AF受体结合的关键残基。显示了对于结合NKG2D.YA(ULBP2.S3,SEQ ID NO.:127)或NKG2D.AF(ULBP2.C，SEQ ID NO.:111；ULBP2.R,SEQ ID NO.:113；ULPB2.AA，SEQ ID NO.:115；和ULBP2.AB，SEQ ID NO.:117)的直写变体所探索的残基R80以及M154-F159区域的位置。
图2：通过尺寸排阻色谱(SEC)测定的候选非天然Fc-eNKG2D变体突变和蛋白聚集特性的总结，如图3和图4中所绘示。
图3：在Akta HiLoad 16/600Superdex 200柱上分析非天然Fc-NKG2D融合蛋白的尺寸排阻色谱比较。正确组装的材料的迁移通过在较高体积下洗脱的离散、对称峰来例示，而聚集的材料在较低体积下洗脱较快。这些修饰的位点和性质通过氨基酸编号Y152、Y199或这两者(从图的顶部开始，SEQ ID NO:48、43、42、58、41和40)来指示。
图4：在Akta Superdex 200 Increase 10/300 GL柱上分析具有一个或两个氨基酸变化的非天然Fc-eNKG2D变体的尺寸排阻色谱图。正确组装的材料的迁移通过在较高体积下洗脱的离散、对称峰来例示，而以在较低体积下洗脱的聚集材料以低幅度宽峰或峰系列为特征。括号中的字母表示分别第152和199位置(由上至下为SEQ ID NO:57、56、55、54、53、52、51、50、49、48、47、46、45、44和40)的氨基酸。
图5：通过结合MICwed-MicAbody或结合至MIC25-MicAbody归一化至野生型NKG2D的eNKG2D变体的百分饱和度(Rmax)。在AHC生物传感器上捕获野生型Fc-NKG2D和每个Fc-eNKG2D受体，然后暴露于20nM的曲妥单抗特异性MicAbody。监测解离动力学并对Fc-eNKG2D融合体的Rmax值进行排序。未测试的样本(nt)是由于SEC分级后严重聚集或表达或回收的材料量不足。
图6：ULBP2野生型、MICwed-和MIC25-利妥昔单抗-MicAbody与Fc-eNKG2D候选物的ELISA结合。图例在图的顶部示出，但由于许多曲线重叠，因此每个图中也标记了单个曲线。
图7：图4示出了Fc-eNKG2D ELISA的EC50值(nM)。nt＝未测试；nb＝即使在300nM时也没有结合或结合非常低，因此不计算EC50值。
图8：eNKG2D变体与野生型配体的结合。将野生型配体(全部为Fc-融合形式)捕获到Octet AHC生物传感器上，并将作为Fc-融合体的每个天然NKG2D、NKG2D.Y152A或eNKG2D5(Y152A/Y199F)从300nM滴定至0.41nM。最大结合应答通过Octet定量。(注意每个图的纵坐标不同)。
图9：通过斑点ELISA验证，相对于天然的NKG2D.wt，ULBP2内的组合突变子集导致噬菌体克隆选择性地与NKG2D.AF结合。根据所选择的噬菌体中出现的频率对突变体进行分级。
图10：各噬菌体变体的滴定ELISA以确认与Fc-NKG2D.AF的选择性结合并减少或消除与Fc-NKG2D.wt的结合。突变详述于图11。
图11：NKG2D.AF选择的ULBP2变体的特异性以利妥昔单抗-MicAbody形式通过定量ELISA保留了其与NKG2D.AF的结合。各ULBP2变体的特定氨基酸修饰显示为其与Fc-NKG2D.wt融合体的结合相对于Fc-NKG2D.AF融合体的结合的比率。ULBP2的氨基酸残基位置是图1B所示者。
图12：在ULBP2.R80W的指示氨基酸位置处的选定突变(图1B；SEQ ID NO:108)产生了Y152A特异性噬菌体克隆。
图13：四种非天然α1-α2ULBP2变体MicAbody与NKG2D.wt、NKG2D.YA和NKG2D.AF结合的ELISA数据。用Fc-NKG2D变体作为捕获剂。用HRP缀合的抗人κ抗体对MicAbody滴定并检测。
图14：使用NetMHC4.0服务器，针对HLA超型代表进行查询，检测ULBP2.C(SEQ IDNO:111)、ULBP2.R(SEQ ID NO:113)、ULBP2.AA(SEQ ID NO:115)和ULBP2.AB(SEQ ID NO:117)的肽-MHCI免疫原性相对于野生型ULBP2(SEQ ID NO:4)的变化。对于输入序列，输入每个变体的可变区(根据图1B中的比对的残基154-159)以及上游和下游9个残基(总共24个残基)，并检测9-mer肽窗口的预测免疫原性。深灰色框对应于强烈预测结合MHCI口袋的肽(定义为％等级<0.5)，因此具有很大的机会被呈递。浅灰色框对应于预测的弱结合剂(％等级<2)。有关详细信息，请参阅实施例5文本。
图15：使用NetMHCII 2.3服务器，针对HLA-DR、HLA-DQ、HLA-DP进行查询，检测ULBP2.C(SEQ ID NO:111)、ULBP2.R(SEQ ID NO:113)、ULBP2.AA(SEQ ID NO:115)和ULBP2.AB(SEQ ID NO:117)的肽-MHC II类免疫原性相对于野生型ULBP2(SEQ ID NO:4)的变化。对于输入序列，输入每个变体的可变区(根据图1B中的比对的残基154-159)以及上游和下游15个残基(总共36个残基)，并检测15-mer肽窗口的预测免疫原性。深灰色框对应于强烈预测结合MHCII口袋并因此可能被呈递和免疫原性的肽。浅灰色框对应于预测的弱结合剂。
图16A和图16B：ELISA测量的，由ULBP2.wt(野生型)、ULBP2.R80W(其对野生型NKG2D具有增强的亲和力)、ULPB2.S3(NKG2D.YA-选择的正交变体)或ULBP2.R(NKG2D.AF-选择的正交变体)组成的，利妥昔单抗-MicAbody，与天然NKG2D-wt、NKG2D-YA和NKG2D-AF的结合。图16A：ELISA曲线。由于TMB-Ultra ELISA开发试剂的沉淀，一些测定中的在较高浓度下458nm吸收的减少是在较高浓度下在高亲和力结合子中经常观察到的假象。图16B：根据图16A中的曲线在GraphPad Prism中确定的EC50值(以nM为单位)。nd＝不确定，因为增加的浓度和结合之间没有关系。
图17A、图17B和图17C：在用未经转导或经NKG2D.wt、NKG2D.YA或NKG2D.AF CAR构建体转导的CD8效应细胞(CD8效应细胞由CD8a铰链/跨膜结构域、细胞内4-1BB和CD3ζ信号域组成)进行的体外细胞裂解测定中。将靶细胞预负载钙黄绿素并以增加的效应子与靶标(E:T)比率暴露于效应细胞。5小时后定量释放的钙黄绿素。图17A：HeLa细胞的细胞裂解。图17B：被转染以过表达表面ULBP1的HeLa细胞的裂解。图17C：表面表达非天然ULBP2.R的HeLa细胞的细胞裂解。误差棒对应于实验中技术复制品的标准偏差。
图18A和图18B：MicAbody通过NKG2D-CAR CD8 T细胞定向的肿瘤株的细胞裂解。图18A：随着ULBP2.S2或ULBP2.R利妥昔单抗-Micabody浓度的增加，表达利妥昔单抗靶标、CD20的Ramos细胞被预加载钙黄绿素，并以20:1的E:T比暴露于NKG2D.AF-或NKG2D.YA-CAR细胞。孵育2小时后定量细胞裂解水平。图18B：将被转染以表达人Her2的小鼠肿瘤系CT26用作与Ramos细胞平行的细胞裂解靶标。用饱和浓度(5nM)的利妥昔单抗-ULBP2.R、曲妥单抗-ULBP2.R或两种MicAbody的等摩尔混合物预先武装NKG2D.AF-CAR CD8 T细胞。通过洗涤除去未结合的MicAbody，并将CD8细胞以两种不同的E:T比率加入靶细胞中。2小时后测量细胞裂解。
具体实施方式
免疫系统的自然杀伤(NK)细胞和某些(CD8+αβ和γδ)T细胞作为对肿瘤细胞和感染细胞的一线天然防御在人类和其他哺乳动物中具有重要作用(Cerwenka,A.,andL.L.Lanier.2001.NK cells,viruses and cancer.Nat.Rev.Immunol.1:41-49)。NK细胞和某些T细胞在其表面显示NKG2D，这是一种突出的同源二聚体表面免疫受体，负责识别靶细胞并激活对病理细胞的天然防御(Lanier,LL,1998.NK cellreceptors.Ann.Rev.Immunol.16:359-393；Houchins JP et al.1991.DNA sequenceanalysis of NKG2,a family of related cDNA clones encoding type II integralmembrane proteins on human NK cells.J.Exp.Med.173:1017-1020；Bauer,S et al.,1999.Activation of NK cells and T cells by NKG2D,a receptor for stress-inducible MICA.Science 285:727-730)。人NKG2D分子具有一个C型凝集素样(胞外)结构域，与其八个不同的同源配体结合，研究最多的配体是具有84％的序列同一性或同源性的单体MICA和MICB，其为主要组织相容性复合物(MHC)I类链相关糖蛋白(MIC)的多态性类似物(Weis et al.1998.The C-type lectin superfamily of the immunesystem.Immunol.Rev.163:19-34；Bahram et al.1994.A second lineage of mammalianMHC class I genes.PNAS 91:6259-6263；Bahram et al.1996a.Nucleotide sequence ofthe human MHC class I MICA gene.Immunogenetics 44:80-81；Bahram and SpiesTA.1996.Nucleotide sequence of human MHC class I MICB cDNA.Immunogenetics 43:230-233)。MICA和MICB的非病理性表达仅限于肠上皮、角质形成细胞、内皮细胞和单核细胞，但这些MIC蛋白的异常表面表达是对增殖、氧化和热休克等多种细胞应激的应答，并标志着细胞的病理性表达(Groh et al.1996.Cell stress-regulated human MHC class Igene expressed in GI epithelium.PNAS 93:12445-12450；Groh etal.1998.Recognition of stress-induced MHC molecules by intestinalγδTcells.Science 279:1737-1740；Zwirner et al.1999.Differential expression ofMICA by endothelial cells,fibroblasts,keratinocytes and monocytes.HumanImmunol.60:323-330)。MIC蛋白的病理性表达似乎也与某些自身免疫性疾病有关(Ravetch,JV and Lanier LL.2000.Immune Inhibitory Receptors.Science 290:84-89；Burgess,SJ.2008.Immunol.Res.40:18-34)。NKG2D配体的不同调控，如多态的MICA和MICB，对于为免疫系统提供一种识别和响应广泛的紧急信号的手段，同时仍然保护健康细胞免受不必要的攻击是重要的(Stephens HA,(2001)MICA and MICB genes:can the enigma oftheir polymorphism be resolved？Trends Immunol.22:378-85；Spies,T.2008.Regulation of NKG2D ligands:a purposeful but delicate affair.NatureImmunol.9:1013-1015)。
病毒感染是MIC蛋白表达的常见诱因，并确定病毒感染的细胞受NK或T细胞攻击(Groh et al.1998；Groh et al.2001.Co-stimulation of CD8+αβT cells by NKG2D viaengagement by MIC induced on virus-infected cells.Nat.Immunol.2:255-260；Cerwenka,A.,and L.L.Lanier.2001)。事实上，为了避免对其宿主细胞的这种攻击，巨细胞病毒和其他病毒已经进化出阻止MIC蛋白在其感染的细胞表面表达的机制，以逃避先天免疫系统的靶向(Lodoen,M.,K.Ogasawara,J.A.Hamerman,H.Arase,J.P.Houchins,E.S.Mocarski,and L.L.Lanier.2003.NKG2D-mediated NK cell protection againstcytomegalovirus is impaired by gp40 modulation of RAE-1molecules.J.Exp.Med.197:1245-1253；Stern-Ginossar et al.,(2007)Host immunesystem gene targeting by viral miRNA.Science 317:376-381；Stern-Ginossar etal.,(2008)Human microRNAs regulate stress-induced immune responses mediatedby the receptor NKG2D.Nature Immunology 9:1065-73；Slavuljica,I A Busche,MBabic,M Mitrovic,I
Figure BDA0002703864890000031
Cekinovic,E Markova Car,EP Pugel,A Cikovic,VJLisnic,WJ Britt,U Koszinowski,M Messerle,A Krmpotic and SJonjic.2010.Recombinant mouse cytomegalovirus expressing a ligand for theNKG2D receptor is attenuated and has improved vaccineproperties.J.Clin.Invest.120:4532-4545)。
MIC蛋白的表达也在许多肿瘤细胞上被诱导，其中MIC蛋白的存在可以使它们对NK细胞的靶向和裂解变得敏感。不足为奇的是，不表达MIC蛋白是许多恶性细胞，如肺癌和成胶质细胞瘤脑癌的一种逃避天然免疫系统的方式(Busche,A et al.2006,NK cellmediated rejection of experimental human lung cancer by genetic overexpression of MHC class I chain-related gene A.Human Gene Therapy 17:135-146；Doubrovina,ES,MM Doubrovin,E Vider,RB Sisson,RJ O’Reilly,B Dupont,and YMVyas,2003.Evasion from NK Cell Immunity by MHC Class I Chain-RelatedMolecules Expressing Colon Adenocarcinoma(2003)J.Immunology 6891-99；Friese,M.et al.2003.MICA/NKG2D-mediated immunogene therapy of experimentalgliomas.Cancer Research 63:8996-9006；Fuertes,MB,MV Girart,LL Molinero,CIDomaica,LE Rossi,MM Barrio,J Mordoh,GA Rabinovich and NW Zwirner.(2008)Intracellular Retention of the NKG2D Ligand MHC Class I Chain-Related Gene Ain Human Melanomas Confers Immune Privilege and Prevents NK Cell-MediatedCytotoxicity.J.Immunology,180:4606-4614)。
已经解决了人MICA与NKG2D结合的高分辨结构，其表明MICA的α3结构域与NKG2D没有直接的相互作用(Li et al.2001.Complex structure of the activatingimmunoreceptor NKG2D and its MHC class I-like ligand MICA.Nature Immunol.2:443-451；Protein Data Bank accession code 1HYR)。与MICB的一样，MICA的α3结构域通过短的、灵活的接头连接到α1-α2平台结构域，并且自身自然地定位为平台和MIC表达细胞表面之间的“间隔序列(spacer)”。人MICA和MICBα3结构域的三维结构几乎相同(94个C-αα′上的均方根距离<
Figure BDA0002703864890000032
)，并且在功能上是可互换的(Holmes et al.2001.StructuralStudies of Allelic Diversity of the MHC Class I Homolog MICB,a Stress-Inducible Ligand for the Activating Immunoreceptor NKG2D.J Immunol.169:1395-1400)。
已描述了某些修饰的非天然NKG2D配体的α1-α2结构域与天然人NKG2D受体结合的亲和力比天然α1-α2结构域更高(Candice S.E.Lengyel,Lindsey J.Willis,PatrickMann,David Baker,Tanja Kortemme,Roland K.Strong and BenjaminJ.McFarland.Mutations Designed to Destabilize the Receptor-Bound ConformationIncrease MICA-NKG2D Association Rate and Affinity.Journal of BiologicalChemistry Vol.282,no.42,pp.30658–30666,2007；Samuel H.Henager,Melissa A.Hale,Nicholas J.Maurice,Erin C.Dunnington,Carter J.Swanson,Megan J.Peterson,JosephJ.Ban,David J.Culpepper,Luke D.Davies,Lisa K.Sanders,and BenjaminJ.McFarland.Combining different design strategies for rational affinitymaturation of the MICA-NKG2D interface.Protein Science 2012VOL21:1396—1402)。此处我们描述了NKG2D配体的非天然α1-α2结构域，其已被修饰以与非天然NKG2D受体结合，这些受体本身在两个特定位点发生突变，其中每一个单独导致其与所有目前已知人NKG2D配体的天然α1-α2结构域的结合受损或失去结合(David J.Culpepper,Michael K.Maddox,Andrew B.Caldwell,and Benjamin J.McFarland.Systematic mutation andthermodynamic analysis of central tyrosine pairs in polyspecific NKG2Dreceptor interactions.(Mol Immunol.2011January；48(4):516–523)。
本发明创造的双特异性分子由(a)特异性修饰的非天然α1-α2结构域和(b)特异性靶向异源分子组成，包括但不限于异源肽或多肽。双特异性分子可结合嵌合抗原受体(CAR)，其中CAR的受体由非天然NKG2D受体胞外域组成，该胞外域以比其天然α1-α2结构域更高的亲和力结合其同源修饰的α1-α2结构域。双特异性分子的第二特异性是由特异性结合其自身的相应靶标分子的异源组分组成。然后，由这类CAR和同源双特异性分子组成的免疫系统的遗传工程化细胞可以克服许多缺点，包括已知的严重全身毒性、抗原逃逸，以及目前CAR-T和CAR-NK细胞疗法的有限和不受控制的持久性(Kalos M,Levine,BL,Porter,DL,Katz,S,Grupp,SA,Bagg,A and June,C..T Cells with chimeric antigen receptorshave potent antitumor effects and can establish memory in patients withadvanced leukemia.Sci Transl Med 2011；3:95ra73；Morgan RA,Yang JC,Kitano M,Dudley ME,Laurencot CM,Rosenberg SA.Case report of a serious adverse eventfollowing the administration of T cells transduced with a chimeric antigenreceptor recognizing ERBB2.Mol Ther 2010,18:843–851)。
可以使用基因转移技术修饰T细胞和NK细胞以在其表面上直接和稳定地表达跨膜信号传导受体，跨膜信号传导受体通过取自抗体的结合结构域赋予T细胞新的抗原特异性(Saar Gill&Carl H.June.Going viral:chimeric antigen receptor T cell therapyfor hematological malignancies.Immunological Reviews 2015.Vol.263:68–89；Wolfgang Glienke,Ruth Esser,Christoph Priesner,Julia D.Suerth,Axel Schambach,Winfried S.Wels,Manuel Grez,Stephan Kloess,Lubomir Arseniev and UlrikeKoehl.2015.Advantages and applications of CAR-expressing natural killercells.Front.Pharmacol.doi:10.3389/fphar.2015.00021)。这种表达嵌合抗原受体的T细胞(CAR-T细胞)是将特异性抗体的抗原识别结构域与CD3-ζ链的胞内结构域结合成单一嵌合蛋白以及来自共刺激分子如CD27、CD28、ICOS、4-1BB或OX40的胞质多肽序列的这种方法的应用，该CD3-ζ链的胞内结构域是来自内源性T细胞抗原受体(TCR)的信号的初级递质。如此构建的CAR可在以类似于内源性T细胞受体但不依赖于主要组织相容性复合体(MHC)的方式结合靶向抗原时触发T细胞活化。
如本文所用的“可溶性MIC蛋白”、“可溶性MICA”和“可溶性MICB”是指含有MIC蛋白的α1、α2和α3结构域但没有跨膜或细胞内结构域的MIC蛋白。NKG2D配体、ULBP1-6天然不具有α3结构域(Cerwenka A,Lanier LL.2004.NKG2D ligands:unconventional MHC classI-like molecules exploited by viruses and cancer.Tissue Antigens 61(5):335–43.doi:10.1034/j.1399-0039.2003.00070.x.PMID 12753652)。NKG2D配体的“α1-α2结构域”是指结合NKG2D受体的配体的蛋白结构域。
在一些实施方案中，本发明的非天然NKG2D配体蛋白的α1-α2结构域与NKG2D配体的原生或天然α1-α2结构域(SEQ ID NO:1-9分别为MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6和OMCP)具有至少80％的同一性或同源性。在其它实施方案中，修饰的α1-α2结构域与NKG2D配体的原生或天然α1-α2结构域具有至少85％的同一性。在其它实施方案中，修饰的α1-α2结构域与天然NKG2D配体蛋白的原生或天然α1-α2结构域具有至少90％的同一性并且结合非天然NKG2D。
优选地，本发明的非天然MIC蛋白的修饰的或非天然的α1-α2结构域与OMPC的原生或天然α1-α2结构域(SEQ ID NO:9)或者八种已知的人NKG2D配体蛋白(SEQ ID NO:1-8)中的一种具有至少80％的同一性或同源性，结合非天然NKG2D胞外域。在一些实施方案中，非天然α1-α2结构域与NKG2D配体蛋白的原生或天然α1-α2结构域具有至少85％的同一性并且结合非天然NKG2D。在其他实施方案中，非天然α1-α2平台结构域与人天然α1-α2结构域蛋白的原生或天然α1-α2平台具有至少90％、95％、96％、97％、98％或99％的同一性并且结合该非天然NKG2D。
在一些实施方案中，异源分子标签可以与可溶性MIC蛋白的非天然α1-α2结构域的N端或C端融合以帮助纯化可溶性MIC蛋白。标签序列包括肽，如聚组氨酸、myc-肽、FLAG标签、链霉亲和素样标签，或小分子如生物素。这种标签可以在通过本领域技术人员已知的方法分离MIC分子后除去。
在本发明的另一些实施方案中，可使NKG2D配体的α1-α2结构域发生特异性突变以产生结合非天然NKG2D受体的非天然α1-α2结构域，其自身经工程改造以对天然NKG2D配体具有降低的亲和力。这可以例如通过基因工程来完成。如此修饰的非天然NKG2D受体可用于在NK细胞，T细胞或免疫系统的其它细胞的表面上产生基于NKG2D的嵌合抗原受体(CAR)，其可优先结合由本发明的非天然α1-α2结构域组成的分子并被其激活。如下所述，与当前的CAR-T细胞和CAR-NK细胞相比，这些非天然NKG2D受体对及其发明的同源非天然NKG2D配体将为治疗癌症和病毒感染提供重要的安全性、功效和制造优势。
用CARS工程化T细胞已经成为癌症过继T细胞疗法的一种很有前景的方法，并且靶向许多不同分子的CARS已经在CAR-T细胞中作为恶性肿瘤的治疗药物进行了测试(PorterDL,Levine BL,Kalos M,Bagg A,June CH.,2011.Chimeric antigen receptor-modifiedT cells in chronic lymphoid leukemia.N Engl J Med.365:725–733.)。虽然在接受表达CD19特异性嵌合抗原受体的T细胞的过继转移的数百名患者中观察到了显著的临床功效，但是常规工程化CAR以靶向特定抗原、从患者分离自体T细胞、基因工程改造自体T细胞以表达个性化CAR、在体外扩增修饰的细胞、以及控制其生产质量，的程序，都较繁琐和昂贵。
一旦自体CAR-T细胞被回输至供体患者，它们在体内的扩张就无法控制，从而产生了一种被称为“活治疗”的治疗，其没有显示与功效的剂量应答关系(Gill和June，2015)。此外，肿瘤从CAR-T细胞中逃逸可能是通过肿瘤细胞上的抗原丢失而发生的(StephanA.Grupp,M.D.,Ph.D.,Michael Kalos,Ph.D.,David Barrett,M.D.,Ph.D.,RichardAplenc,M.D.,Ph.D.,David L.Porter,M.D.,Susan R.Rheingold,M.D.,David T.Teachey,M.D.,Anne Chew,Ph.D.,Bernd Hauck,Ph.D.,J.Fraser Wright,Ph.D.,MichaelC.Milone,M.D.,Ph.D.,Bruce L.Levine,Ph.D.,and Carl H.June,M.D.Chimeric AntigenReceptor–Modified T Cells for Acute Lymphoid Leukemia.N.Engl.J.Med.2013；368:1509–1518)。虽然这种逃逸途径可以通过不同靶点的CAR-T细胞的序贯治疗或通过初始输注含有两种或两种以上特异性CAR的T细胞产品来解决，但这样的步骤代表着制造工艺、质量控制和成本方面的显著额外扩增。
除了以具有单链抗体结合结构域(scFv)的肿瘤为靶标的CAR-T细胞外，利用NKG2D受体的配体结合结构域的CAR-T细胞已经在动物和最近在人类中进行了研究(Sentman CL,Meehan KR.NKG2D CARs as cell therapy for cancer.Cancer J.2014Mar-Apr；20(2):156-9.doi:10.1097/PPO.0000000000000029；Manfred Lehner,Gabriel
Figure BDA0002703864890000051
Julia Proff,Niels Schaft,Jan
Figure BDA0002703864890000052
Florian Full,Armin Ensser,Yves A.Muller,AdelheidCerwenka,Hinrich Abken,Ornella Parolini,Peter F.Ambros,Heinrich Kovar,Wolfgang Holter.Redirecting T Cells to Ewing's Sarcoma Family of Tumors by aChimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNATransfection.Research Article|published 15Feb 2012|PLOS ONE 10.1371/journal.pone.0031210；www.clinicaltrials.gov NCT02203825)。由于NKG2D配体在应激细胞(如肿瘤细胞)表面的表达增加，因此此天然NKG2D配体家族作为癌症免疫治疗的靶点是很有意义的(Spear P,Wu MR,Sentman ML,Sentman CL.NKG2D ligands as therapeutictargets.Cancer Immun.2013May 1；13:8.；Song DG,Ye Q,Santoro S,Fang C,Best A,Powell DJ Jr.,Chimeric NKG2D CAR-expressing T cell-mediated attack of humanovarian cancer is enhanced by histone deacetylase inhibition.Hum GeneTher.2013Mar；24(3):295-305)。一个NKG2D CAR是全长NKG2D受体与CD3ζ(NKG2Dζ)的融合体；另一个是只将NKG2D的胞外域与由CD28的跨膜和胞内结构域与CD3ζ的信号结构域(NKG2D28ζ)组成的第二代CAR支架融合。由于NKG2D的激活依赖于DAP10的存在，因此还构建了DAP10与NKG2Dζ(NKG2Dζ10)共表达的CAR-T细胞。表达任何上述NKG2D CAR的T细胞响应于NKG2D配体刺激而产生IFNγ和TNFα，并且在体外有效地杀死表达NKG2D配体的肿瘤靶标(Heather VanSeggelen,Joanne A.Hammill,Anna Dvorkin-Gheva,Daniela G.M.Tantalo,Jacek M.Kwiecien,Galina F.Denisova,Brian Rabinovich,Yonghong Wan,JonathanL.Bramson,T cells engineered with chimeric antigen receptors targeting NKG2Dligands display lethal toxicity in mice,Molecular Therapy 2015Oct；23(20):1600-10；doi:10.1038/mt.2015.119)。NK细胞对广谱肿瘤亚型的细胞毒活性也可通过表达基于NKG2D-DAP10-CD3ζ的CAR显著增强(Yu-Hsiang Chang,John Connolly,NorikoShimasaki,Kousaku Mimura,Koji Kono,and Dario Campana.Chimeric Receptor withNKG2D Specificity Enhances Natural Killer Cell Activation and Killing ofTumor Cells.Cancer Res；73(6)March 15,2013)。
然而，在注入同源鼠宿主后，与天然NKG2D受体的天然配体结合并被其激活的这些CAR-T构建体出现显著毒性。与未处理的对照小鼠相比，在用基于NKG2D的CAR-T细胞处理的有瘤和无瘤小鼠中观察到毒性迹象，包括身体条件差、弓腰姿势、呼吸费力和核心体温降低。NKG2D CAR-T细胞毒性严重程度不同，其中NKG2Dζ10细胞毒性严重，NKG2D28ζ细胞毒性中等，NKG2Dζ细胞毒性可耐受。当小鼠在过继转移表达任何NKG2D CAR的T细胞之前接受化疗时，毒性临床症状和死亡率恶化(VanSeggelen等人，2015)。已知化疗和放射在其它健康组织上诱导NKG2D配体(Xiulong Xu,Geetha S Rao,Veronika Groh,Thomas Spies,PaoloGattuso,Howard L Kaufman,Janet Plate and Richard A Prinz.Majorhistocompatibility complex class I-related chain A/B(MICA/B)expression intumor tissue and serum of pancreatic cancer:Role of uric acid accumulation ingemcitabine-induced MICA/B expression.BMC Cancer 2011,11:194.doi:10.1186/1471-2407-11-194；GannagéM,Buzyn A,Bogiatzi SI,Lambert M,Soumelis V,DalCortivo L,Cavazzana-Calvo M,Brousse N,Caillat-Zucman Induction of NKG2Dligands by gamma radiation and tumor necrosis factor-alpha may participate inthe tissue damage during acute graft-versus-hostdisease.Transplantation.2008Mar 27；85(6):911-5.doi:10.1097/TP.0b013e31816691ef.)。进一步的表征显示，毒性与全身细胞因子风暴和肺部致死性炎症水平相吻合。这些数据警示当使用天然NKG2D配体进行靶向免疫治疗时必须格外小心，并证明靶向这些配体的CAR的T细胞表达——特别是强活化的CAR——在体内可能是有害的(VanSeggelen等人，2015)。
由非天然NKG2D受体的胞外域组成的CAR-T或CAR-NK细胞不与或几乎不与天然NKG2D配体结合，不受限于上述活化形式，因此不会像表达基于天然NKG2D受体的CAR的细胞那样具有毒性。此外，细胞上非天然NKG2D受体的胞外区不会受可溶性天然NKG2D配体或髓源性抑制细胞(MDSC)的下调(Deng W,Gowen BG,Zhang L,Wang L,Lau S,Iannello A,XuJ,Rovis TL,Xiong N,Raulet DH,2015.Antitumor immunity.A shed NKG2D ligand thatpromotes natural killer cell activation and tumor rejection.Science.2015Apr3；348(6230):136-9.doi:10.1126/science.1258867.Epub 2015Mar 5)。然而，当具有非天然NKG2D受体的胞外域的此类CAR细胞通过双特异性分子与本发明的同源非天然α1-α2结构域及其已发现并结合其预期靶标的异源靶向基序结合时，CAR将被活化并且表达CAR-细胞的效应子功能。
因为除了在由同源的非天然α1-α2结构域组成的结合的双特异性分子存在的情况下，由非天然NKG2D受体胞外域组成的CAR-T或CAR-NK细胞的细胞因子释放和细胞裂解活性不被引发，其中双特异性分子也已经结合其靶标阵列，它们的活化可以通过所施用的双特异性分子来控制，其作为生物药物将表现出本领域熟知的药代动力学和药效学。在发生不良事件的情况下，医师可以简单地改变所施用的双特异性分子的给药方案，而不是如目前所进行的那样必须采用诱导的自杀机制以破坏输注的CAR细胞(Monica Casucci andAttilio Bondanza.Suicide Gene Therapy to Increase the Safety of ChimericAntigen Receptor-Redirected T Lymphocytes.J Cancer.2011；2:378–382)。此外，这样的具有不同特异性靶向基序的双特异性分子可以同时或顺序施用以帮助解决由于靶抗原丢失导致的肿瘤抗性和逃逸，而不必创构、扩增和输注多个不同的自体同源CAR细胞(Gill和June，2015)。由于所有CAR构建对于所有CAR细胞可以是相同的，并且靶向特异性仅由所施用的本发明的双特异性分子的靶向基序确定，因此CAR-细胞制造过程将被简化并且更便宜。
作为用于附着至NKG2D配体的非天然α1-α2结构域的候选物的亲本或受体蛋白或多肽的实例包括但不限于抗体，由Ig折叠或Ig结构域组成的蛋白，包括募集天然分子或未能募集或结合天然分子的修饰的Fc结构域、球蛋白、白蛋白、纤连蛋白和纤连蛋白结构域、整合素、荧光蛋白、酶、外膜蛋白、受体蛋白、T细胞受体、嵌合抗原受体、病毒抗原、病毒衣壳、细胞受体的病毒配体、组蛋白、激素、细胞因子和修饰的细胞因子如白介素、扭结菌素(knottin)、环肽或多肽、主要组织相容性(MHC)家族蛋白、MIC蛋白、凝集素和凝集素的配体。还可以将非蛋白分子如多糖、树枝状大分子、聚二醇、肽聚糖、抗生素和聚酮化合物连接至NKG2D配体的修饰的α1-α2结构域。
因此，本发明扩展了这种显著的，非常有前景的用CAR-T细胞、CAR-NK细胞和CAR-巨噬细胞样细胞控制癌症的免疫学方法的多样性和实用性，同时克服了许多这些目前公认的困难。
如本文所用，“肽”、“多肽”和“蛋白”可互换使用；和“异源分子”、“异源肽”、“异源序列”或“异源原子”分别是非天然或在与主题分子物理结合时通常发现的分子、肽、核酸或氨基酸序列或原子。如本文所用，“非天然的”和“修饰的”可互换使用。如本文所用，“天然的”、“原生的”和“野生型”可互换使用，“NKG2D”和“NKG2D受体”可互换使用。本文中的术语“抗体”以最广泛的含义使用，并且具体涵盖单克隆抗体、多特异性抗体(例如双特异性抗体)和抗体片段，只要它们表现出所需的生物学活性即可。“抗体片段”包括抗体的一部分，优选包括其抗原结合区。抗体片段的实例包括Fab、Fab′、F(ab′)2、Fv片段和可插入的Fv's；双抗体；线性抗体；单链抗体分子；和由抗体片段形成的多特异性抗体。“抗体片段”还可以包括抗体的Fc部分。
可与“包含”、“含有”或“其特征在于”互换使用的术语“包括”是包括性的或开放性的语言，并且不排除另外的、未列举的元件或方法步骤。短语“由……组成(consistingof)”排除权利要求中未指明的任何元件、步骤或成分。短语“基本上由……组成(consisting essentially of)”将权利要求的范围限制为特定的材料或步骤，以及那些不会实质上影响要求保护的发明的基本和新颖特征的材料或步骤。本公开考虑了与这些短语中的每一个的范围相对应的本发明的组合物和方法的实施方案。因此，包括所述要素或步骤的组合物或方法考虑了特定实施方案，其中所述组合物或方法基本上由这些要素或步骤组成或由这些要素或步骤组成。
本文引用的所有参考文献通过引用以其整体并入本文，无论先前是否具体并入。如本文所用，术语“一(a)”、“一(an)”和“任何(any)”各自旨在包括单数和复数形式两者。
现在已经充分描述了本发明，本领域技术人员将理解，在不偏离本发明的精神和范围的情况下并且在没有过度实验的情况下，可以在宽范围的等价参数、浓度和条件内进行相同的操作。虽然已经结合本发明的具体实施例描述了本发明，但是应当理解，本发明能够进一步修改。本申请旨在涵盖大体上遵循本发明的原理的本发明的任何变化、用途或适应，并且包括在本发明所属领域内的已知或惯用实践内的与本公开内容的偏离，并且可应用于上文阐述的基本特征。
实施例
修饰的NKG2D受体胞外域和NKG2D配体修饰的α1-α2结构域
实施例1：将人NKG2D受体的酪氨酸152修饰为丙氨酸(Y152A)、酪氨酸199修饰为苯丙氨酸(Y199F)，以创构惰性NKG2D胞外域
其他人已经证明，人NKG2D的酪氨酸152或199位的突变，相当于NKG2D胞外域的第75位和第122位(图1A，SEQ ID NO:17)可以极大地减少与天然配体MICA的结合(DavidJ.Culpepper,Michael K.Maddox,Andrew B.Caldwell,and BenjaminJ.McFarland.Systematic mutation and thermodynamic analysis of centraltyrosine pairs in polyspecific NKG2D receptor interactions.MolImmunol.2011January；48(4):516–523)。我们推测，虽然任一酪氨酸残基的突变极大地影响了NKG2D与其天然配体的结合能力，但酪氨酸152(Y152)和酪氨酸199(Y199)处的同时突变实际上会消除受体与所有天然配体结合的能力。因此，我们寻求探索单个的和组合的Y152和Y199取代，并对它们的生物化学行为进行表征，目的是鉴定不能与任何天然配体结合的单突变体和双突变体变体。那些表达和装配良好的变体是特别令人感兴趣的，因为这些有意义的惰性配体可以更容易地产生以用于分析。
天然NKG2D(野生型)胞外域(NKG2D.wt，SEQ ID NO:17)和候选非天然NKG2D变体胞外域(SEQ ID NO:18-35)——也称为“工程NKG2D”或“eNKG2D”通过短因子Xa可识别的Ile-Glu-Gly-Arg接头(SEQ ID NO:38)将其作为与人IgG1 Fc(无Fab结构域)的C端的融合体克隆，并且可互换地称为Fc-NKG2D.wt或NKG2D.wt和Fc-eNKG2D或eNKG2D(SEQ ID NO:40-58)。合成了对应于MHCI信号序列(SEQ ID NO:36和37)、带有接头的人IgG1 Fc(SEQ ID NO:39)和NKG2D胞外域变体(SEQ ID NO:59-77)的
Figure BDA0002703864890000071
DNA片段(加州圣地亚哥Integrated DNATechnologies)，并将其插入pD2610-V12(加州纽瓦克市ATUM)。探索在Y152、Y199或Y152/Y199组合突变处取代的DNA构建体在Expi293TM细胞(马萨诸塞州沃尔瑟姆的赛默飞世尔科技公司)中瞬时表达(图2)，并通过蛋白A亲和层析(伊利诺伊州罗克福德的PierceBiotechnology，货号20334)纯化分泌的蛋白。通过尺寸排阻色谱(SEC)在Akta PurSuperdex柱上对洗脱的物质进行表征，并且将正确组装的，尺寸合适的物质进行分级，并且从聚集峰中分离出来，然后纳入分析。
纯化的NKG2D.Y199A-Fc融合体的SEC表征揭示了主要聚集的物质的组成(图3)。相比之下，天然Fc-NKG2D融合体和Fc-NKG2D.Y152A融合材料均通过离散的非聚集峰区分，该峰容易与更快速迁移的聚集体区分。在Y152A/Y199A双突变体Fc-NKG2D融合变体中，Y199A突变对聚集的影响也是明显的，表明其对蛋白错误折叠具有显著影响(图3)。因此，在NKG2D变体中包括具有Y152突变的任何组合的Y199A的这个方面对于生产后续工程工作所必需的材料提出了挑战并且提出了关于在细胞表面上组装和呈递的考虑。因此，人们努力探索Y152和Y199处的其他取代，这些取代可以结合在一起产生更强大的分子。eNKG2D组合的Y152和Y199突变体候选物作为Fc融合体进行了检测，并在(图2)中进行了详细说明。此外，通过SEC分析所有纯化和表达的Fc-eNKG2D融合候选物，并且它们的色谱图显示了不同水平的聚集体形成(图2-4)。在152位残基丙氨酸、丝氨酸、苏氨酸和缬氨酸处探索的单个氨基酸取代均未影响Fc-NKG2D分子的组装，尽管Y152-亮氨酸(Y152L)产生高度聚集的物质。与丙氨酸相似，199位的谷氨酸和天冬氨酸都不能被耐受，尽管苯丙氨酸仅是度地增加了聚集体的形成。在所研究的突变组合中，Y152A/Y199F、Y152S/Y199F、Y152T/Y199F和Y152F/Y199F没有负面地影响期望的二聚体形成，而其他组合会导致聚集的增加(图2-4)。
实施例2：用非天然NKG2D配体变体产生基于抗体的双特异性分子“MicAbody”
产生融合到人IgG1的非天然MicA变体，编码例如MICwed(SEQ ID NO:79)和MIC25(SEQ ID NO:81)的α1-α2个结构域的DNA多核苷酸用引物进行PCR扩增，引物还引入了编码用于融合到人IgG1的C端κ轻链(SEQ ID NO:84)的APTSSSGGGGS接头或用于融合到人IgG1的C端重链(SEQ ID NO:82)的GGGS接头的多核苷酸。此外，在重链的CH2结构域引入了两个突变——D265A/N297A(KABAT编号)——这两个突变减少了与所有FcγR受体的结合，从而消除了抗体依赖的细胞毒性(ADCC)功能。(Shields et al.,2001JBC,276:6591-6604)。编码没有GPI-连接(SEQ ID NO:12)的野生型ULBP2(ULBP2.wt)的α1-α2结构域的多核苷酸被类似地克隆，并将其与编码接头和IgG1重链或轻链的DNA多核苷酸融合。这些双特异性抗体，单数称为“MicAbodyTM”，复数称为“MicAbody”，对融合的α1-α2结构域为二价。为了探索eNKG2D工程目的而用于生成MicAbody的抗体的实例包括但不限于曲妥单抗(SEQ ID NO:94和96)和利妥昔单抗(SEQ ID NO:98和100)以及随后分别称为“曲妥单抗-MicAbody”(例如SEQ ID NO:102和104)和“利妥昔单抗-MicAbody”(例如SEQ ID NO:106)。通过Gibson克隆(马萨诸塞州伊普斯威奇的新英格兰生物技术公司(New England Biolabs Inc.))将融合构建体单独地插入pD2610-V12(加州纽瓦克的ATUM)中。对于识别特定抗原的给定抗体，将编码重链的质粒和编码轻链的质粒与天然或非天然NKG2D配体融合，共转染以在Expi293TM细胞(马萨诸塞州沃尔瑟姆的赛默飞世尔科技公司)中瞬时表达。或者，将编码与天然或非天然NKG2D配体融合的重链的质粒和用于轻链的质粒共转染。分泌的双特异性抗体通过蛋白A亲和层析(伊利诺伊州罗克福德的Pierce Biotechnology，货号20334)纯化，洗脱的材料通过尺寸排阻色谱法(SEC)在Akta Pur Superdex柱上表征，并根据需要进行分级。此外，对纯化的样本进行SDS-PAGE分析以验证融合的重链和融合的轻链物种的预期分子量。
实施例3：鉴定不能与天然NKG2D结合配体或与野生型NKG2D结合增强的非天然配体结合的修饰NK2GD变体
利用平板ELISA方法分析α1-α2变异体与天然(野生型)NKG2D和非天然eNKG2D蛋白的胞外域的结合亲和力。将每个SEC分级的天然Fc-NKG2D和非天然Fc-eNKG2D融合体以磷酸盐缓冲盐水(PBS)中1μg/mL的包被浓度4℃过夜包被Nunc Maxisorp96孔板(马萨诸塞州沃尔瑟姆的赛默飞世尔科技公司)的单独的孔上。在20至22℃用PBS/0.05％吐温-20(PBS-T)洗涤3次，在20至22℃用PBS中0.5％牛血清白蛋白(PBS-B)封闭2小时。在PBS/0.5％牛血清白蛋白(BSA)/0.05％吐温-20(PBS-BT)中，针对平板结合的天然或非天然Fc-NKG2D融合体在20至22℃滴定MicAbody 60分钟，用PBS-T在20至22℃洗涤3次，用PBS-BT(马萨诸塞州剑桥的Abcam)中的HRP缀合抗人κ检测结合的双特异性蛋白，并用1-StepTMUltra TMB ELISA底物溶液(马萨诸塞州沃尔瑟姆的赛默飞世尔科技公司)显色。ULBP2.wt利妥昔单抗-MicAbody(SEQ ID NO:98和106)的结合区分野生型NKG2D和eNKG2D变体，但与后者的结合减少，并且配体变体——MICwed(SEQ ID NO:96和102)和MIC25(SEQ ID NO:96和104)——在鉴定与消除的配体结合的eNKG2D变体时更严格。每个eNKG2D变体与所有三种双特异性配体的结合行为表明，NKG2D修饰的组合导致野生型和变体配体的结合最大程度地降低，并使铅惰性NKG2D变体得以选择。
还使用FortéBio Octet系统(均为加利福尼亚州弗里蒙特的FortéBio LLC)通过Bio-Layer Interferometry(BLI)对eNKG2D变体与配体的结合进行了额外的生物物理分析。对于这些实验，人NKG2D配体MICA-Fc、MICB-Fc、ULBP1-Fc、ULBP2-Fc、ULBP3-Fc和ULBP4-Fc购自明尼苏达州明尼阿波利斯的R&D Systems公司。在抗人IgG Fc捕获(AHC)生物传感器尖端上捕获MicAbody形式的配体。基线建立后，尖端暴露于300nM至0.41nM的Fc-eNKG2D融合蛋白滴定系列中，并监测PBS-BT中所有步骤的结合/解离动力学。随后，将Fc-eNKG2D融合蛋白捕获到AHC尖端上，并滴定MicAbody以表征结合动力学。
为了测定通过天然NKG2D与MICwed或MIC25的结合所定义的最大应答，将天然Fc-NKG2D融合体捕获到AHC生物传感器上，并且将20nM曲妥单抗-MICwed或20nM曲妥单抗-MIC25 MicAbody孵育2分钟，然后观察解离动力学30秒。然后用Fc-eNKG2D融合受体作为捕获剂在相同条件下进行结合分析，并且将每个eNKG2D的结合水平列为由Fc-NKG2D.wt确定的最大结合应答的百分比(图5)。对于MICwed，除了Y199F外，所有单突变体Fc-eNKG2D变体的应答减少至50％。Y199F保持100％结合应答。然而，所有的双突变体Fc-eNKG2D变体完全消除了与MICwed的结合。对于MIC25，所有单突变体Fc-eNKG2D变体和Y152V/Y199F相对于野生型Fc-NKG2D结合维持100％结合应答。然而，与几种双突变体Fc-eNKG2D变体(包括Y152A/Y199F、Y152S/Y199F和Y152T/Y199F)的结合降低至50％。
用作为捕获剂的Fc-eNKG2D融合体进行ELISA测定，其中ULBP2.wt，MICwed，MIC25MicAbody从300nM开始滴定(图6)。当可能时，使用GraphPad Prism(图7中)计算EC50值。天然NKG2D与基于ULBP2、MICwed和MIC25的MicAbody结合，亲和力计算为Kds值分别为1.4、0.007和0.005nM。虽然ULBP2和MICwed MicAbody与所有单个突变体eNKG2D候选物的亲和力降低，但MIC25与eNKG2D候选物的结合保持不变。然而，所有双突变体eNKG2D候选物都以Micabody形式消除或显著减少了与所有三种配体——ULBP2、MICwed和MIC25——的结合。
通过Octet分析和ELISA，eNKG2D变体eNKG2D5(Y152A/Y199F)、eNKG2D7(Y152S/Y199F)、eNKG2D8(Y152T/Y199F)和eNKG2D9(Y152V/Y199F)降低或消除了与基于ULBP2、MICwed和MIC25的MicAbody的结合(图5和7)。此外，eNKG2D 5、7和8的聚集量最少，这表明在293T表达上有更强大的蛋白组装(图2)。更仔细地检测eNKG2D5(SEQ ID NO:48)与野生型配体的结合，因为在Octet AHC尖端捕捉到的是MicAbody。与天然(SEQ ID NO:40)NKG2D相比，单个突变体Fc-NKG2D.Y152A(SEQ ID NO:41)与所有天然配体的结合减少(图8)。与Y152AeNKG2D相比，eNKG2D5(Y152A/Y199F)应答反应曲线进一步降低。eNKG2D5(Y152A/Y199F，以下简称为“AF”或“NKG2D.AF”)被选为设计同源选择性、正交、非天然配体的主要NKG2D变体。
实施例4：构建与非天然NKG2D.AF胞外域选择性结合的正交非天然α1-α2结构域
我们利用噬菌体展示技术设计与Fc-NKG2D AF(SEQ ID NO:48)受体选择性结合的正交非天然α1-α2结构域。作为起点，选择与天然野生型NKG2D(NKG2D.wt)胞外域具有高亲和力的非天然ULBP2.R80Wα1-α2结构域(图1B；SEQ ID NO:108)作为亲本结构域以进一步通过噬菌体展示进行诱变和筛选。为ULBP2.R80W的α1-α2结构域(SEQ ID NO:108)生成了合成DNA文库，其另外还有C8S突变，以消除二硫键的可能性。配体上处于结合状态的氨基酸残基的密码子定位在天然NKG2D受体上的Y152和Y199位附近，被NNK密码子取代；文库由154-159位的NNK密码子组成(图1B；SEQ ID NO:110)。将文库克隆为M13噬菌体的pIII次要外壳蛋白的融合体，按照标准方法在SS320大肠杆菌(E.coli)中制备显示诱变的α1-α2结构域突变体的噬菌体颗粒(Andris-Widhopf,J.,Steinberger,P.,Fuller,R.,Rader,C.,and Barbas,C.F.,3rd.(2011))。在非生物素化的天然Fc-NKG2D.wt竞争蛋白存在的情况下，通过选择性地捕获与生物素化的Fc-NKG2D.AF蛋白结合的噬菌体克隆，对这些α1-α2噬菌体展示文库进行了筛选，以获得与非天然NKG2D.AF受体高度结合的亲和力。用递增浓度的非生物素化天然Fc-NKG2D通过多轮竞争选择循环来富集选择性克隆。
经过4轮选择后，将噬菌体克隆分别以96孔的形式排列，进行斑点ELISA以验证与平板结合的非天然NKG2D.AF相对于NKG2D.wt的优选差异结合。用生物素化的M13噬菌体外壳蛋白单克隆抗体E1(马萨诸塞州沃尔瑟姆的赛默飞世尔科技公司)、链霉亲和素-HRP检测(明尼苏达州明尼阿波利斯的R&D Systems)和1-Step Ultra TMB ELISA开发(马萨诸塞州沃尔瑟姆的赛默飞世尔科技公司)检测结合的噬菌体。将每个克隆的斑点ELISA信号表示为噬菌体结合NKG2D.AF与噬菌体结合NKG2D.wt的比率。对比例大于或等于14的那些噬菌体进行测序以鉴定NNK诱变区域内的特异性突变。图9显示了选择性结合NKG2D.AF的每个α1-α2噬菌体变体的选择的氨基酸残基。在鉴定代表相同序列的多个克隆的情况下，绘制ELISA信号的比率，并且通过数据点的聚类验证噬菌体克隆的一致性(数据未显示)。
在ELISA中鉴定的30种变体在单独的单一培养物中扩增以产生高效价的噬菌体微批次。将纯化的噬菌体浓度归一化至OD268＝0.5，然后针对平板结合的Fc-NKG2D.AF或Fc-NKG2D.wt进行1:3稀释系列，噬菌体检测和ELISA开发如上所述进行。以这种方式测定的所有30种变体一致地显示与NKG2D.AF的选择性结合，与NKG2D.wt几乎没有或没有结合(图10)，即使在测定噬菌体的最高浓度下。选择的噬菌体也显示出噬菌体浓度的两个或多个对数的偏移，以实现NKG2D.AF和NKG2D.wt之间的半数最大结合。
为了证实NKG2D.AF选择性α1-α2结构域变体在抗体融合体中保留了特异性结合特性，将21个变异体(图11；例如SEQ ID NO:111-118)克隆为具有利妥昔单抗轻链的APTSSSGGGGS接头的C端融合体(SEQ ID NO:119-126)。通过Gibson克隆(马萨诸塞州伊普斯威奇的新英格兰生物实验室公司(New England Biolabs Inc.))将融合体克隆到哺乳动物表达载体pD2610-V12(ATUM，纽瓦克，加利福尼亚)中，并与亲本抗体的重链(SEQ ID NO:99)共表达为成对的全IgG抗体。根据制造商的方案在Expi293TM细胞(马萨诸塞州沃尔瑟姆的赛默飞世尔科技公司)中进行瞬时表达，并使用标准蛋白-A亲和层析(伊利诺伊州罗克福德的Pierce Biotechnology，货号20334)。ELISA测量每个变体ULBP2α1-α2抗体融合体与非天然Fc-NKG2D.AF和天然Fc-NKG2D.wt的结合，表明它们与NKG2D.AF的结合亲和力明显高于天然NKG2D.wt(图11)。总之，这些数据证明了非天然正交α1-α2结构域的发明，该结构域具有与非天然NKG2D.AF受体结合的高亲和力，并显著降低了与天然NKG2D型受体的结合亲和力。此外，这些正交的α1-α2结构域与抗体多肽的融合体保持了它们的选择性结合特性，并例如在嵌合抗原受体(CAR)T细胞的情况下用于将非天然NKG2D.AF受体重定向到特异性抗原。
实施例5：通过选择性地结合一个或另一个，识别能够区分非天然NKG2D受体变体的非天然NKG2D配体
如上所述，以非天然ULBP2.R80Wα1-α2结构域(SEQ ID NO:108)为起点，进行噬菌体展示以设计与NKG2D.Y152A(以下简称NKG2D.YA)受体选择性结合的正交非天然α1-α2结构域。通过在非生物素化的天然Fc-NKG2D.wt(SEQ ID NO:40)竞争蛋白的存在下选择性捕获与生物素化的Fc-NKG2D.YA(SEQ ID NO:41)蛋白结合的噬菌体克隆，针对与非天然Fc-NKG2D.YA受体的高结合亲和力淘选α1-α2噬菌体展示文库。其他的噬菌体克隆验证工作导致鉴定出相对于Fc-NKG2D.wt优先结合Fc-NKG2D.YA的变体(图12)。例如，ULBP2.S3(SEQ IDNO:127)与天然NKG2D.wt相比，通过ELISA和Octet分析(既呈单体His标签化又呈双特异性抗体融合形式)一致地证明了与非天然NKG2D.YA的选择性结合。这代表了非天然正交α1-α2结构域的发明的一种独特形式，其具有与非天然NKG2D受体(在本例中为NKG2D.YA而不是实施例4中的NKG2D.AF)的高亲和力结合。此外，正交的α1-α2结构域与抗体多肽的融合体保留了它们的选择性结合特性，并用于选择性地将非天然的NKG2D受体重定向到由融合的异源多肽(如抗体)确定的特定分子。
为了确定与NKG2D.YA选择性结合的非天然α1-α2结构域(ULBP2.S3，SEQ ID NO:127)和与NKG2D.AF选择性结合的非天然α1-α2结构域是否能够区分这两种非天然受体变体，进行滴定ELISA。直接比较了结合NKG2D.AF的所有21种选择的α1-α2变体与NKG2D.AF和NKG2D.YA的结合。其中4个变体显示不能结合NKG2D.wt，与NKG2D.AF有很强的亲和力，与NKG2D.AF相比，与NKG2D.YA的结合大大减少(15-20倍)或消除(图13)。还使用NetMHC4.0Server检测了这四个非天然ULBP2α1-α2变体——ULBP2.C,ULBP2.R,ULBP2.AA和ULBP2.AB(SEQ ID NO:111、113、115和117)——相对于野生型ULBP2肽序列(SEQ ID NO:4)的预测免疫原性分布的变化(用9-mer肽分析对所有HLA超型代表的多肽-MHC I类结合进行查询；http://www.cbs.dtu.dk/services/NetMHC/)和NetMHCII 2.3 Server(用15-mer肽分析查询HLA-DR、HLA-DQ、HLA-DP单倍型的多肽-MHC II类结合；http://www.cbs.dtu.dk/services/NetMHCII/)，两者均为丹麦技术大学(Technical University of Denmark)开发的算法(http://www.bioinformatics.dtu.dk/；Andreatta M and Nielsen M,Gappedsequence alignment using artificial neural networks:application to the MHCclass I system,2016 Bioinformatics,32:511,PMID:26515819；Jensen KK,AndreattaM,Marcatili P,Buus S,Greenbaum JA,Yan Z,Sette A,Peters B,and Nielsen M,Improved methods for predicting peptide binding affinity to MHC class Imolecules,2018Immunology,PMID:29315598)。ULBP2.C、ULBP2.R和ULBP2.AB中的突变没有增加预测的免疫原性，而ULPB2.AA的突变在少数单倍型中略有增加(图14和15)。由于ULBP2.R对NKG2D.AF的特异性和缺乏可预测的免疫原性，选择ULBP2.R进行进一步的ELISA分析，以直接将其与ULBP2.S3(NKG2D.YA选择的非天然正交配体)、ULBP2.R80W(与野生型NKG2D亲和力增强的非天然配体)和野生型ULBP2(ULBP2.wt)的结合行为进行比较。相对于野生型NKG2D(NKG2D.wt)和两种惰性、非天然变体NKG2D.YA和NKG2D.AF，测定4种利妥昔单抗-MicAbody试剂(SEQ ID NO:98和121、98和129、131和100以及98和106分别作为ULBP2.R、ULBP2.S3、ULBP2.R80W和ULBP2.wt的重链和轻链)的结合(图16A和16B)。数据表明，NKG2D.YA选择的变体ULBP2.S3作为MicAbody与NKG2D.YA有较高的亲和力，但不与NKG2D.AF或天然NKG2D结合。此外，NKG2D.AF选择的变体ULBP2.R以MicAbody形式与NKG2D.AF以高亲和力结合，但不与NKG2D.YA或天然NKG2D结合。这些结果显示了探索NKG2D-MIC配体轴和开发独特的成对新颖的、选择性的非天然NKG2D受体及其各自的同源非天然MIC配体结合配偶体的巨大潜力。
实施例6：表达非天然NKG2D.AF胞外域的CAR-T细胞的靶向和杀伤活性受融合到异源靶向多肽的正交α1-α2结构域控制
减轻毒性并提高抗肿瘤功效后，选择性控制CAR-T细胞疗法的手段非常受欢迎(Gill和June，同上)。先前已尝试使用CD16的胞外域开发CAR，CD16的胞外域可接着通过治疗性单克隆抗体的Fc结构域结合，从而允许基于抗体的CAR-T靶向控制(Chang等人，同上)。然而，基于CD16的CAR-T细胞可以识别血液和组织中几乎所有的内源性抗体分子，并且用于控制这些细胞的治疗性抗体将遇到来自NK细胞、PMN、单核细胞和巨噬细胞上的内源性CD16受体的竞争。这两个特征分别造成了非肿瘤毒性和较差的药代动力学问题。
天然的NKG2D配体存在于某些健康组织和许多应激组织上，使用当前的NKG2D CAR方法会产生极大的毒性风险(VanSeggelen等人，2015)。Y152A非天然NKG2D受体与非天然α1-α2结构域NKG2D配体特异性结合，构成使用由发明的NKG2D配体的非天然α1-α2结构域组成的双特异性蛋白选择性控制非天然NKG2D CAR活性的手段的实例。
我们设计了具有受体的CAR-T细胞，该受体由修饰的NKG2D的Y152A/Y199F(“AF”)胞外域组成，其与所有天然的NKG2D配体或先前描述的与Y152A修饰的NKG2D(NKG2D.YA)正交和同源的非天然α1-α2结构域缺乏结合。本发明的同源非天然α1-α2结构域与非天然NKG2D.AF胞外域以高亲和力结合，避免与天然NKG2DYA胞外域和NKG2D.YA胞外域结合。因此，与天然NKG2D和非天然NKG2D.YA相比，对非天然NKG2D.AF胞外域表现出较强选择性的工程化α1-α2结构域代表了用于选择性控制非天然NKG2D CAR受体或融合到非天然NKG2D胞外域的任何受体或蛋白质的理想系统，这些受体或蛋白质可以被本发明的非天然α1-α2结构域选择性地结合。本发明进一步使单个细胞能够表达两种不同的CAR——一种由NKG2D.YA组成，另一种由NKG2D.AF组成——每个信号传导具有明显不同的细胞内结构域。这些不同的CAR将通过细胞外暴露于各自的，同源的正交MicAbody或另一种非抗体融合多肽而具有细胞活性的独立、双重控制。
为了证明对采用展示非天然NKG2D.AF胞外域的嵌合受体构建的CAR-T细胞的选择性控制，在前人工作的基础上，我们利用4-1BB/CD3ζ CAR构建体(Campana专利8,399,645)将相应的NKG2D胞外域融合至CAR的CD8铰链区(SEQ ID NO:151、153、155)构建了天然NKG2D.wt(SEQ ID NO:135)、非天然NKG2D.YA(SEQ ID NO:137)或非天然NKG2D.AF(SEQ IDNO:139)胞外域的CAR。将这些构建体(SEQ ID NO:152、154、156)克隆到慢病毒载体中并使用慢病毒转导在原代人CD8阳性T细胞中表达。HeLa细胞在其表面上具有组成型上调水平的MIC配体，包括MICA、MICB、ULBP3和ULBP2/5/6(用于确定这一点的抗体不能区分这三种ULBP；明尼苏达州明尼阿波利斯的R&D系统的人ULBP-2/5/6抗体)。转染HeLa细胞使其表面也过表达天然ULBP1或NKG2D.AF选择的变体ULBP2.R，并将这些细胞用作体外杀伤测定的靶标。用钙黄绿素预负载HeLa靶细胞，并以增加效应子与靶标(E:T)的比率使其暴露于NKG2D.wt-CAR、NKG2D.YA-CAR或NKG2D.AF-CAR CD8细胞5小时，之后将释放到上清液中的钙黄绿素的量进行定量，并归一化为洗涤剂处理后释放的总钙黄绿素(图17A-17C)。由于在HeLa细胞表面天然表达的MIC配体水平升高，表达天然NKG2D(NKG2D.wt)的CD8细胞作为CAR通过这种过表达的天然配体与HeLa细胞结合并实现细胞裂解。然而，NKG2D.YA-和NKG2D.AF-CAR转导的CD8细胞甚至在高E:T比率(与未转导的CD8 T细胞同等水平的活性水平)下显示出极少的天然HeLa细胞裂解。当ULBP1在HeLa细胞表面过表达时，仅NKG2D.wt-CAR CD8 T细胞显著裂解它们。NKG2D.YA-CAR细胞在高E:T比时有一些额外的杀伤作用，但NKG2D.AF-CAR细胞不存在这种情况，表明双突变Y152A/Y199F使NKG2D比单一Y152A突变更有惰性。在过表达NKG2D.AF选择性非天然ULBP2.R的HeLa细胞中，NKG2D.wt-CAR细胞直接裂解(由于内源性MIC配体的识别)，而NKG2D.AF-CAR细胞定向的裂解水平与受体及其选择性配体的结合一致。
为了证明NKG2D.YA-或NKG2D.AF-CAR细胞的裂解只能通过适当的、同源的靶向MicAbody进行，将Ramos细胞与基于利妥昔单抗的MicAbody联合用作细胞裂解的靶标，基于利妥昔单抗的MicAbody与非天然ULBP2.S3或ULBP2.R正交配体连接。如图18A所示，利妥昔单抗-ULBP2.S3 MicAbody可以指导NKG2D.YA-CAR CD8细胞，而不是NKG2D.AF-CAR细胞的细胞杀伤活性，而利妥昔单抗-ULBP2.R MicAbody可以指导NKG2D.AF-CAR，而不是NKG2D.YA-CAR细胞的活性。这进一步证明了两种非天然ULBP2变体对于它们被设计为优选配偶体的同源的非天然NKG2D变体的选择性。为了证明MicAbody抗体部分的特异性，将NKG2D.AF-CARCD8细胞分别与利妥昔单抗-ULBP2.R、曲妥单抗-ULPB2.R(分别为SEQ ID NO:95和133，重链和轻链)或两者在饱和总浓度下等摩尔组合孵育而预先武装，进行体外杀伤测定。在通过洗涤除去未结合的MicAbody后，将CD8细胞应用于已预负载钙黄绿素的Ramos细胞(表达CD20，利妥昔单抗的靶标)或CT26-Her2(经转染以表达人Her2的小鼠细胞系)。在两种不同的E:T比率下孵育2小时后，定量所释放的钙黄绿素的量。如图18B所示，当细胞用利妥昔单抗-MicAbody预先武装时，仅Ramos细胞裂解，而曲妥单抗-MicAbody仅针对CT26-Her2细胞定向细胞裂解活性。然而，当NKG2D.AF-CAR CD8细胞同时用利妥昔单抗和曲妥单抗-ULBP2.RMicAbody预先武装时，两种靶细胞系被裂解，证明这些CAR细胞(借助于已经在受体和配体之间设计的选择性的，优先的配对)容易被复用并且由此被定向以同时结合不同的肿瘤靶标。
序列表
<110> 修普霍斯生物科学有限公司（XYPHOS BIOSCIENCES INC.）
<120> 结合非天然NKG2D受体的非天然NKG2D配体的修饰的α1-α2结构域
<130> F246240
<150> US 62/648,636
<151> 2018-03-27
<160> 156
<170> PatentIn version 3.5
<210> 1
<211> 183
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 MICA α1-α2 结构域
<400> 1
Ala Ala Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp
1 5 10 15
Asp Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly
20 25 30
Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln
35 40 45
Gly Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu
50 55 60
Thr Arg Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala
65 70 75 80
His Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg
85 90 95
Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe
100 105 110
Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu
115 120 125
Trp Thr Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val
130 135 140
Arg Asn Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His
145 150 155 160
Ala Met His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser
165 170 175
Gly Val Val Leu Arg Arg Thr
180
<210> 2
<211> 182
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 MICB α1-α2 结构域
<400> 2
Ala Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp
1 5 10 15
Glu Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln
20 25 30
Pro Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly
35 40 45
Gln Trp Ala Glu Asp Val Leu Gly Ala Lys Thr Trp Asp Thr Glu Thr
50 55 60
Glu Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His
65 70 75 80
Ile Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val
85 90 95
Cys Glu Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr
100 105 110
Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser
115 120 125
Thr Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr
130 135 140
Asn Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala
145 150 155 160
Met Gln Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly
165 170 175
Val Ala Ile Arg Arg Thr
180
<210> 3
<211> 187
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 ULBP1 α1-α2 结构域
<400> 3
Ala Ala Glu Pro His Cys Leu Cys Tyr Asp Phe Ile Ile Thr Pro Lys
1 5 10 15
Ser Arg Pro Glu Pro Gln Trp Cys Glu Val Gln Gly Leu Val Asp Glu
20 25 30
Arg Pro Phe Leu His Tyr Asp Cys Val Asn His Lys Ala Lys Ala Phe
35 40 45
Ala Ser Leu Gly Lys Lys Val Asn Val Thr Lys Thr Trp Glu Glu Gln
50 55 60
Thr Glu Thr Leu Arg Asp Val Val Asp Phe Leu Lys Gly Gln Leu Leu
65 70 75 80
Asp Ile Gln Val Glu Asn Leu Ile Pro Ile Glu Pro Leu Thr Leu Gln
85 90 95
Ala Arg Met Ser Cys Glu His Glu Ala His Gly His Gly Arg Gly Ser
100 105 110
Trp Gln Phe Leu Phe Asn Gly Gln Lys Phe Leu Leu Phe Asp Ser Asn
115 120 125
Asn Arg Lys Trp Thr Ala Leu His Pro Gly Ala Lys Lys Met Thr Glu
130 135 140
Lys Trp Glu Lys Asn Arg Asp Val Thr Met Phe Phe Gln Lys Ile Ser
145 150 155 160
Leu Gly Asp Cys Lys Met Trp Leu Glu Glu Phe Leu Met Tyr Trp Glu
165 170 175
Gln Met Leu Asp Pro Thr Lys Pro Pro Met Val
180 185
<210> 4
<211> 189
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 ULBP2 α1-α2 结构域
<400> 4
Ala Ala Glu Pro His Ser Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys
1 5 10 15
Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu
20 25 30
Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val
35 40 45
Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln
50 55 60
Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Arg
65 70 75 80
Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln
85 90 95
Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser
100 105 110
Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu
115 120 125
Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu
130 135 140
Lys Trp Glu Asn Asp Lys Val Val Ala Met Ser Phe His Tyr Phe Ser
145 150 155 160
Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp
165 170 175
Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro Pro Met Val
180 185
<210> 5
<211> 187
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 ULBP3 α1-α2 结构域
<400> 5
Ala Ala Glu Pro His Ser Leu Trp Tyr Asn Phe Thr Ile Ile His Leu
1 5 10 15
Pro Arg His Gly Gln Gln Trp Cys Glu Val Gln Ser Gln Val Asp Gln
20 25 30
Lys Asn Phe Leu Ser Tyr Asp Cys Gly Ser Asp Lys Val Leu Ser Met
35 40 45
Gly His Leu Glu Glu Gln Leu Tyr Ala Thr Asp Ala Trp Gly Lys Gln
50 55 60
Leu Glu Met Leu Arg Glu Val Gly Gln Arg Leu Arg Leu Glu Leu Ala
65 70 75 80
Asp Thr Glu Leu Glu Asp Phe Thr Pro Ser Gly Pro Leu Thr Leu Gln
85 90 95
Val Arg Met Ser Cys Glu Cys Glu Ala Asp Gly Tyr Ile Arg Gly Ser
100 105 110
Trp Gln Phe Ser Phe Asp Gly Arg Lys Phe Leu Leu Phe Asp Ser Asn
115 120 125
Asn Arg Lys Trp Thr Val Val His Ala Gly Ala Arg Arg Met Lys Glu
130 135 140
Lys Trp Glu Lys Asp Ser Gly Leu Thr Thr Phe Phe Lys Met Val Ser
145 150 155 160
Met Arg Asp Cys Lys Ser Trp Leu Arg Asp Phe Leu Met His Arg Lys
165 170 175
Lys Arg Leu Glu Pro Thr Ala Pro Pro Met Val
180 185
<210> 6
<211> 187
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 ULBP4 α1-α2 结构域
<400> 6
Ala Ala Glu Pro His Ser Leu Cys Phe Asn Phe Thr Ile Lys Ser Leu
1 5 10 15
Ser Arg Pro Gly Gln Pro Trp Cys Glu Ala Gln Val Phe Leu Asn Lys
20 25 30
Asn Leu Phe Leu Gln Tyr Asn Ser Asp Asn Asn Met Val Lys Pro Leu
35 40 45
Gly Leu Leu Gly Lys Lys Val Tyr Ala Thr Ser Thr Trp Gly Glu Leu
50 55 60
Thr Gln Thr Leu Gly Glu Val Gly Arg Asp Leu Arg Met Leu Leu Cys
65 70 75 80
Asp Ile Lys Pro Gln Ile Lys Thr Ser Asp Pro Ser Thr Leu Gln Val
85 90 95
Glu Met Phe Cys Gln Arg Glu Ala Glu Arg Cys Thr Gly Ala Ser Trp
100 105 110
Gln Phe Ala Thr Asn Gly Glu Lys Ser Leu Leu Phe Asp Ala Met Asn
115 120 125
Met Thr Trp Thr Val Ile Asn His Glu Ala Ser Lys Ile Lys Glu Thr
130 135 140
Trp Lys Lys Asp Arg Gly Leu Glu Lys Tyr Phe Arg Lys Leu Ser Lys
145 150 155 160
Gly Asp Cys Asp His Trp Leu Arg Glu Phe Leu Gly His Trp Glu Ala
165 170 175
Met Pro Glu Pro Thr Val Ser Pro Pro Met Val
180 185
<210> 7
<211> 198
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 ULBP5 α1-α2 结构域
<400> 7
Gly Leu Ala Asp Pro His Ser Leu Cys Tyr Asp Ile Thr Val Ile Pro
1 5 10 15
Lys Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp
20 25 30
Glu Lys Thr Phe Leu His Tyr Asp Cys Gly Ser Lys Thr Val Thr Pro
35 40 45
Val Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala
50 55 60
Gln Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu
65 70 75 80
Leu Asp Ile Gln Leu Glu Asn Tyr Ile Pro Lys Glu Pro Leu Thr Leu
85 90 95
Gln Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Gly Ser Gly
100 105 110
Ser Trp Gln Leu Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser
115 120 125
Glu Asn Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys
130 135 140
Glu Lys Trp Glu Asn Asp Lys Asp Met Thr Met Ser Phe His Tyr Ile
145 150 155 160
Ser Met Gly Asp Cys Thr Gly Trp Leu Glu Asp Phe Leu Met Gly Met
165 170 175
Asp Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro Pro Thr Met Ser Ser
180 185 190
Gly Thr Ala Gln Pro Arg
195
<210> 8
<211> 189
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 ULBP6 α1-α2 结构域
<400> 8
Ala Ala Glu Pro His Ser Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys
1 5 10 15
Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu
20 25 30
Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val
35 40 45
Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln
50 55 60
Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Leu
65 70 75 80
Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln
85 90 95
Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser
100 105 110
Trp Gln Phe Ser Ile Asp Gly Gln Thr Phe Leu Leu Phe Asp Ser Glu
115 120 125
Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu
130 135 140
Lys Trp Glu Asn Asp Lys Asp Val Ala Met Ser Phe His Tyr Ile Ser
145 150 155 160
Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp
165 170 175
Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro Pro Met Val
180 185
<210> 9
<211> 161
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 OMCP α1-α2 结构域
<400> 9
Ala Ala Ala Glu Pro His Lys Leu Ala Phe Asn Phe Asn Leu Glu Ile
1 5 10 15
Asn Gly Ser Asp Thr His Ser Thr Val Asp Val Tyr Leu Asp Asp Ser
20 25 30
Gln Ile Ile Thr Phe Asp Gly Lys Asp Ile Arg Pro Thr Ile Pro Phe
35 40 45
Met Ile Gly Asp Glu Ile Phe Leu Pro Phe Tyr Lys Asn Val Phe Ser
50 55 60
Glu Phe Phe Ser Leu Phe Arg Arg Val Pro Thr Ser Thr Pro Tyr Glu
65 70 75 80
Asp Leu Thr Tyr Phe Tyr Glu Cys Asp Tyr Thr Asp Asn Lys Ser Thr
85 90 95
Phe Asp Gln Phe Tyr Leu Tyr Asn Gly Glu Glu Tyr Thr Val Lys Thr
100 105 110
Gln Glu Ala Thr Asn Lys Asn Met Trp Leu Thr Thr Ser Glu Phe Arg
115 120 125
Leu Lys Lys Trp Phe Asp Gly Glu Asp Cys Ile Met His Leu Arg Ser
130 135 140
Leu Val Arg Lys Met Glu Asp Ser Lys Arg Arg Thr Val Pro Pro Met
145 150 155 160
Val
<210> 10
<211> 543
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 MICA α1-α2 结构域
<400> 10
gagccccaca gtcttcgtta taacctcacg gtgctgtcct gggatggatc tgtgcagtca 60
gggtttctca ctgaggtaca tctggatggt cagcccttcc tgcgctgtga caggcagaaa 120
tgcagggcaa agccccaggg acagtgggca gaagatgtcc tgggaaataa gacatgggac 180
agagagacca gagacttgac agggaacgga aaggacctca ggatgaccct ggctcatatc 240
aaggaccaga aagaaggctt gcattccctc caggagatta gggtctgtga gatccatgaa 300
gacaacagca ccaggagctc ccagcatttc tactacgatg gggagctctt cctctcccaa 360
aacctggaga ctaaggaatg gacaatgccc cagtcctcca gagctcagac cttggccatg 420
aacgtcagga atttcttgaa ggaagatgcc atgaagacca agacacacta tcacgctatg 480
catgcagact gcctgcagga actacggcga tatctaaaat ccggcgtagt cctgaggaga 540
aca 543
<210> 11
<211> 561
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULBP1 α1-α2 结构域
<400> 11
gctgctgagc cccactgtct ctgctacgac tttattataa ctcctaagtc aagaccagag 60
cctcagtggt gcgaagtaca aggtttggtt gacgaaaggc ctttccttca ctacgattgt 120
gtgaaccata aggcaaaggc tttcgccagc ctgggtaaga aggtaaacgt tactaagacg 180
tgggaggagc agacggaaac cctccgtgat gtggttgact ttcttaaggg tcagctcctc 240
gatatccaag tggagaattt aatccctatc gaaccgctca ctctgcaggc cagaatgtca 300
tgcgaacatg aagcacacgg tcatggaaga ggtagttggc aatttttatt taacggtcaa 360
aaattcctgc tgttcgactc aaacaaccgc aaatggactg cgctgcaccc tggagctaag 420
aagatgactg aaaaatggga gaagaacaga gacgttacca tgttcttcca gaagatttcc 480
ctgggagatt gtaagatgtg gttagaggag ttcttaatgt actgggaaca gatgctggac 540
cccacaaaac cccccatggt g 561
<210> 12
<211> 567
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULBP2 α1-α2 结构域
<400> 12
gctgctgagc cccatagtct gtgttacgac atcacagtta ttcccaagtt caggcccgga 60
ccgcgctggt gtgccgtgca aggacaagtc gacgaaaaaa cctttcttca ttacgattgc 120
ggaaataaga ctgtaacgcc agtctctcct ttaggtaaga agttaaacgt cactacggcg 180
tggaaggcac aaaaccccgt cctgcgcgag gtcgtcgaca tcctgactga acaattgcgc 240
gacatccagc tcgagaatta cactccaaag gagcctctta ccctgcaggc tagaatgtct 300
tgcgagcaaa aggcagaggg ccactcctcc ggcagctggc agttcagttt cgacggacaa 360
atctttctgt tattcgattc agagaagaga atgtggacta cagttcaccc cggtgcccgt 420
aaaatgaagg agaagtggga aaacgacaaa gtggtggcga tgtcattcca ctatttctcg 480
atgggagact gcatcggttg gctggaagat ttcctcatgg gtatggactc cactttggag 540
ccatcggctg gtgccccccc catggtg 567
<210> 13
<211> 561
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULBP3 α1-α2 结构域
<400> 13
gctgctgagc cccacagctt gtggtacaac ttcaccatta tccacttgcc gagacatggc 60
cagcagtggt gcgaagtgca atcgcaagtc gaccaaaaaa acttcttatc atacgactgc 120
ggcagcgata aggtcttatc tatgggtcat ttggaggaac agctctacgc gaccgacgcc 180
tggggtaaac agctcgagat gctccgtgag gttggacaga ggctgagact ggaactggct 240
gacactgagc tggaagattt cacacctagt ggtccactca cattgcaagt acgcatgagc 300
tgcgagtgtg aggccgatgg atacattagg ggcagctggc agtttagctt cgacggaagg 360
aaattcctgc tcttcgacag taacaatagg aagtggactg ttgtgcatgc tggtgcgcgc 420
agaatgaagg aaaagtggga gaaagatagc ggcctgacga ccttcttcaa gatggtgtct 480
atgcgtgact gtaagagctg gctcagagat ttcctcatgc atcgcaagaa gaggttagaa 540
cctaccgctc cccccatggt g 561
<210> 14
<211> 561
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULBP4 α1-α2 结构域
<400> 14
gctgctgagc cccactctct ttgcttcaac ttcaccatta aatccctgag caggcctggt 60
cagccgtggt gtgaggcgca ggtctttctt aacaagaatc tcttcctcca atacaactct 120
gataacaaca tggtaaagcc actgggtctc ctgggtaaaa aagtctatgc tacgagcact 180
tggggagaac tcacccagac tcttggcgag gtaggaagag acctgcgcat gctcctctgc 240
gatataaagc cccaaattaa gaccagtgat ccgtccactt tacaagtcga aatgttctgc 300
caaagggagg ctgaacgctg caccggagcc tcttggcagt tcgcgaccaa tggcgaaaag 360
tccctcttgt tcgatgccat gaatatgacc tggaccgtga tcaatcatga ggcctctaag 420
atcaaggaga cgtggaaaaa ggaccgcggc cttgaaaagt actttaggaa gttgtctaaa 480
ggagactgcg accattggtt acgcgagttc ctcggccatt gggaagcgat gcccgagcca 540
acggttagcc cccccatggt g 561
<210> 15
<211> 567
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULBP6 α1-α2 结构域
<400> 15
gctgctgagc cccactcctt atgctatgat atcaccgtga ttccaaagtt ccgaccagga 60
ccccgatggt gcgccgtaca gggacaggtc gacgaaaaga cttttttaca ttacgactgc 120
ggtaacaaga cagtcacacc ggtaagtcct ttgggaaaaa agttaaacgt aaccactgct 180
tggaaggccc agaaccccgt ccttcgagaa gtagtggata ttttgactga acagctgctt 240
gacatccagc tggaaaacta cacacccaaa gagcccctga ctcttcaagc gcgtatgtcg 300
tgtgagcaaa aggccgaagg acacagctcc ggatcctggc agttcagtat cgacggtcag 360
accttcctcc tcttcgattc agaaaagcgc atgtggacta ctgtgcaccc cggcgctcgt 420
aagatgaagg aaaagtggga gaatgataag gacgttgcca tgagttttca ttacattagt 480
atgggagatt gcatcggttg gctggaagac ttcctgatgg gtatggatag tacccttgaa 540
cctagtgccg gagctccccc catggtg 567
<210> 16
<211> 483
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 OMCP α1-α2 结构域
<400> 16
gctgctgctg agccccacaa gcttgcgttc aacttcaatc tggaaataaa cggttcagat 60
acccattcaa ccgtggacgt ttatttagac gattcgcaga taatcacctt tgacggcaag 120
gacatccgcc caactatccc gttcatgata ggtgacgaaa tcttccttcc tttttataag 180
aatgtgttct ctgagttctt cagtttgttc cgccgcgtcc ctacctcaac cccctacgaa 240
gacttgactt atttctatga atgcgactac accgacaaca aatctacatt cgatcaattc 300
tacctgtaca acggtgaaga gtacaccgtg aagactcaag aggctactaa caagaacatg 360
tggctgacca cttccgagtt cagactgaag aagtggttcg acggcgagga ctgtatcatg 420
caccttagaa gtttagtgag gaaaatggaa gatagcaaga gaagaacagt gccccccatg 480
gtg 483
<210> 17
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 天然 NKG2D 胞外域
<400> 17
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 18
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152A 胞外域
<400> 18
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 19
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y199A 胞外域
<400> 19
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Ala Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 20
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152A/Y199A 胞外域
<400> 20
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Ala Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 21
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y199F eNKG2D1 胞外域
<400> 21
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 22
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152S eNKG2D2 胞外域
<400> 22
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ser His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 23
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152T eNKG2D3 胞外域
<400> 23
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Thr His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 24
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152V eNKG2D4 胞外域
<400> 24
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Val His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 25
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152A/Y199F eNKG2D5 胞外域
<400> 25
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 26
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152L/Y199F eNKG2D6 胞外域
<400> 26
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Leu His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 27
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152S/Y199F eNKG2D7 胞外域
<400> 27
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ser His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 28
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152T/Y199F eNKG2D8 胞外域
<400> 28
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Thr His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 29
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152V/Y199F eNKG2D9 胞外域
<400> 29
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Val His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 30
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y199D eNKG2D10 胞外域
<400> 30
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Asp Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 31
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y199E eNKG2D11 胞外域
<400> 31
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Glu Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 32
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152D/Y199D eNKG2D12 胞外域
<400> 32
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Asp His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Asp Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 33
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152E/Y199E eNKG2D13 胞外域
<400> 33
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Glu His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Glu Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 34
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152L eNKG2D14 胞外域
<400> 34
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Leu His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 35
<211> 139
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 非天然 NKG2D Y152F/Y199F eNKG2D15 胞外域
<400> 35
Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu
1 5 10 15
Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
20 25 30
Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala
35 40 45
Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu
50 55 60
Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Phe His Trp Met Gly Leu
65 70 75 80
Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile
85 90 95
Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
100 105 110
Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr
115 120 125
Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 36
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 MHCI 信号序列
<400> 36
Met Gly Leu Gly Pro Val Phe Leu Leu Leu Ala Gly Ile Phe Pro Phe
1 5 10 15
Ala Pro Pro Gly Ala Ala Ala
20
<210> 37
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 MHCI 信号序列
<400> 37
atgggccttg gcccagtgtt tctgctgttg gcaggcattt tcccttttgc tccgcccggc 60
gccgcagcc 69
<210> 38
<211> 237
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc 带 IEGR 接头
<400> 38
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg
225 230 235
<210> 39
<211> 711
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc 带 IEGR 接头
<400> 39
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg c 711
<210> 40
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D 融合体
<400> 40
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 41
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152A 融合体
<400> 41
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 42
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y199A 胞外域
<400> 42
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Ala Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 43
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152A/Y199A 胞外域
<400> 43
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Ala Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 44
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y199F eNKG2D1 融合体
<400> 44
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 45
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152S eNKG2D2 融合体
<400> 45
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 46
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152T eNKG2D3 融合体
<400> 46
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Thr His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 47
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152V eNKG2D4 融合体
<400> 47
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Val His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 48
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152A/Y199F eNKG2D5 融合体
<400> 48
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 49
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152L/Y199F eNKG2D6 融合体
<400> 49
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Leu His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 50
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152S/Y199F eNKG2D7 融合体
<400> 50
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Ser His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 51
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152T/Y199F eNKG2D8 融合体
<400> 51
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Thr His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 52
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152V/Y199F eNKG2D9 融合体
<400> 52
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Val His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 53
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y199D eNKG2D10 融合体
<400> 53
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Asp Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 54
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y199E eNKG2D11 融合体
<400> 54
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Glu Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 55
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152D/Y199D eNKG2D12 融合体
<400> 55
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Asp His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Asp Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 56
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152E/Y199E eNKG2D13 融合体
<400> 56
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Glu His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Glu Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 57
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152L eNKG2D14 融合体
<400> 57
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Leu His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 58
<211> 376
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 Fc-NKG2D Y152F/Y199F eNKG2D15 融合体
<400> 58
Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn
225 230 235 240
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
245 250 255
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
260 265 270
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
275 280 285
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
290 295 300
Leu Leu Lys Leu Val Lys Ser Phe His Trp Met Gly Leu Val His Ile
305 310 315 320
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
325 330 335
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
340 345 350
Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr
355 360 365
Tyr Ile Cys Met Gln Arg Thr Val
370 375
<210> 59
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D 融合体
<400> 59
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcatatcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 60
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152A 融合体
<400> 60
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcagctcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 61
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y199A 胞外域
<400> 61
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcataccatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgctata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 62
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152A/Y199A 胞外域
<400> 62
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcagctcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgctata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 63
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y199F eNKG2D1 融合体
<400> 63
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcataccatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 64
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152S eNKG2D2 融合体
<400> 64
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcataccatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 65
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152T eNKG2D3 融合体
<400> 65
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcaactcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 66
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152V eNKG2D4 融合体
<400> 66
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcagtgcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 67
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152A/Y199F eNKG2D5 融合体
<400> 67
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcagctcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 68
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152L/Y199F eNKG2D6 融合体
<400> 68
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcactgcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 69
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152S/Y199F eNKG2D7 融合体
<400> 69
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcaagtcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 70
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152T/Y199F eNKG2D8 融合体
<400> 70
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcaactcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 71
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152V/Y199F eNKG2D9 融合体
<400> 71
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcagtgcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 72
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y199D eNKG2D10 融合体
<400> 72
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcatatcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgatata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 73
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y199E eNKG2D11 融合体
<400> 73
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcataccatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgagata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 74
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152D/Y199D eNKG2D12 融合体
<400> 74
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcagatcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgatata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 75
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152E/Y199E eNKG2D13 融合体
<400> 75
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcagagcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgagata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 76
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152L eNKG2D14 融合体
<400> 76
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcactgcatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 77
<211> 1128
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人 IgG1 Fc-NKG2D Y152F/Y199F eNKG2D15 融合体
<400> 77
atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720
tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780
aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840
tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900
gaggaccagg atttacttaa actggtgaag tcattccatt ggatgggact agtacacatt 960
ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020
ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080
gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128
<210> 78
<211> 181
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 MICwed α1-α2
<400> 78
Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly
1 5 10 15
Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro
20 25 30
Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln
35 40 45
Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg
50 55 60
Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile
65 70 75 80
Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys
85 90 95
Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr
100 105 110
Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr
115 120 125
Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn
130 135 140
Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met
145 150 155 160
His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val
165 170 175
Val Leu Arg Arg Thr
180
<210> 79
<211> 543
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 MICwed α1-α2
<400> 79
gagcctcaca gcctccggta taatttgact gtactctctt gggatggctc cgtgcagtcc 60
ggctttctga ctgaagttca tctcgacggt caacctttcc tgcgctgcga ccgacaaaaa 120
tgccgcgcca agccccaagg gcagtgggcc gaagatgtac tgggaaacaa gacctgggac 180
cgggagacac gagacctgac aggctggggc aaggacttgc gcatgacact cgcccatatc 240
aaggaccaga aggaaggatt gcactctttg caagagattc gcgtgtgtga aatccacgag 300
gacaattcaa cgaggagctc ccagcacttc tattacgatg gagaactctt cttgtcacag 360
aacttggaaa ccaaggaatg gactatgcct cagagctctc gggcacagac tctcgctatg 420
aacgttagaa acttccttaa ggaggatgct atggagaccg atactcacta ccacgccatg 480
cacgccgact gcctccagga actgcggaga tatctgaagt ccggcgtggt tttgagaaga 540
acc 543
<210> 80
<211> 181
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 MIC25 α1-α2
<400> 80
Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly
1 5 10 15
Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro
20 25 30
Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln
35 40 45
Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg
50 55 60
Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile
65 70 75 80
Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys
85 90 95
Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr
100 105 110
Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr
115 120 125
Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn
130 135 140
Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met
145 150 155 160
Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val
165 170 175
Val Leu Arg Arg Thr
180
<210> 81
<211> 543
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 MIC25 α1-α2
<400> 81
gagcctcaca gcctccggta taatttgact gtactctctt gggatggctc cgtgcagtcc 60
ggctttctga ctgaagttca tctcgacggt caacctttcc tgcgctgcga ccgacaaaaa 120
tgccgcgcca agccccaagg gcagtgggcc gaagatgtac tgggaaacaa gacctgggac 180
cgggagacac gagacctgac aggctggggc aaggacttgc gcatgacact cgcccatatc 240
aaggaccaga aggaaggatt gcactctttg caagagattc gcgtgtgtga aatccacgag 300
gacaattcaa cgaggagctc ccagcacttc tattacgatg gagaactctt cttgtcacag 360
aacttggaaa ccctcgaatg gactatgcct cagagctctc gggcacagac tctcgctatg 420
aacgttagaa acttccttaa ggaggatgct atggagaccg atactcacta ccacgccatg 480
cgcgccgact gcctctctga actgcggaga tatctgaagt ccggcgtggt tttgagaaga 540
acc 543
<210> 82
<211> 330
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人 IgG1 重链 CH1-CH2-CH3 D265A/N297A
<400> 82
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 83
<211> 990
<212> DNA
<213> 人工序列
<220>
<223> 人 IgG1 重链 CH1-CH2-CH3 D265A/N297A
<400> 83
gcgtcgacca agggcccgtc agtgttcccg ctggccccgt catccaagtc cacgtctggg 60
ggcacagcag ccctgggatg cttggtcaag gactacttcc ccgagcccgt gactgtgtcc 120
tggaactccg gagcactgac ctccggagtg cacacctttc ccgcggtgct gcagtcctcc 180
ggactgtact ccctgtcgtc ggtcgtgacc gtgccgagct cctcgctcgg aacccagacc 240
tacatctgca acgtgaacca caagccctcg aacaccaaag tggacaagaa ggtcgagccc 300
aaaagctgcg acaagactca cacttgtccg ccgtgccccg cccccgaact gctgggtggc 360
ccctccgtgt tcctgttccc gcctaagcct aaggacaccc ttatgatcag ccgcacccct 420
gaagtgacct gtgtcgtcgt ggcagtgtca cacgaggacc cggaggtcaa gttcaattgg 480
tacgtggacg gcgtggaagt gcataacgca aagaccaagc ctcgggagga acagtacgcc 540
tcgacctacc gcgtggtgtc agtcctgact gtgctgcacc aggactggct gaacgggaag 600
gagtacaagt gcaaagtgtc gaacaaggcc ctgccggctc caattgaaaa gaccatcagc 660
aaggccaagg gccagccaag ggaaccacag gtgtacaccc tccctccttc ccgggacgag 720
ctgaccaaaa accaagtgtc cctgacttgc cttgtgaagg ggttctaccc ttctgacatt 780
gccgtcgaat gggaatcgaa cggacagcct gaaaacaact ataagactac cccgcccgtg 840
ctggattccg acggaagctt cttcctgtac tccaagctga ccgtggacaa gtcgagatgg 900
cagcagggaa atgtgttcag ctgctccgtg atgcatgagg cgctgcacaa ccactacacc 960
cagaagtcac tgagcctctc ccccggaaag 990
<210> 84
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 人κ轻链
<400> 84
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 85
<211> 321
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 人κ轻链
<400> 85
agaaccgtgg ccgccccgag cgtgttcatt ttccctccct ccgacgagca gttgaaatcg 60
ggcaccgcta gcgtggtctg ccttctcaac aatttctatc cacgggaagc caaagtgcag 120
tggaaggtcg acaacgcgct ccaatccggg aactcacagg aatccgtgac tgagcaggat 180
tccaaggact cgacctactc cctgtcatcc acgctgaccc tgagcaaggc agactacgag 240
aagcacaagg tctacgcctg cgaagtgaca caccagggac tgtccagccc cgtgaccaag 300
agcttcaaca gaggagaatg c 321
<210> 86
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 曲妥珠单抗 VH
<400> 86
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 87
<211> 360
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 曲妥珠单抗 VH
<400> 87
gaagtccaat tggtcgaatc aggcggtgga ctcgtgcaac ctggaggttc gttacgctta 60
tcatgtgctg caagtggatt taatattaaa gatacctaca tccactgggt acgtcaagct 120
cccggcaagg gtctcgagtg ggtcgcacgc atttacccca ccaacggata cacgcgctac 180
gccgattcag tgaagggacg tttcacaatc tctgctgata ctagcaaaaa taccgcatac 240
ctccagatga actctcttag ggccgaggac acagctgtgt actactgtag ccgttgggga 300
ggagacggtt tttacgcaat ggattactgg ggccaaggaa ccctggtcac agtttcatcg 360
<210> 88
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 曲妥珠单抗 VL
<400> 88
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 89
<211> 321
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 曲妥珠单抗 VL
<400> 89
gatatccaaa tgactcaatc accatcttca ctctccgcga gcgtgggtga tcgggtcacc 60
atcacatgta gggcgagcca agatgtgaat accgccgtcg cgtggtatca acaaaagccg 120
ggaaaagcac caaaactgct tatatactct gcatccttcc tgtactctgg ggtgccaagc 180
cggttctccg gtagtagatc tggtactgac tttacactca ctatcagcag tctgcaacct 240
gaggactttg cgacatacta ttgccagcag cactacacaa ccccacctac atttggtcag 300
gggacaaagg tggagatcaa g 321
<210> 90
<211> 121
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 利妥昔单抗 VH
<400> 90
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110
Ala Gly Thr Thr Val Thr Val Ser Ala
115 120
<210> 91
<211> 363
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 利妥昔单抗 VH
<400> 91
caagttcagc ttcagcagcc gggggctgag ttggtgaaac ccggggccag tgtgaagatg 60
agctgtaaag cgagcggcta caccttcact tcttataata tgcattgggt taagcaaacg 120
ccaggaaggg ggctggagtg gatcggcgct atttacccag gtaacggtga cacatcatat 180
aaccaaaagt ttaagggaaa ggcaaccctc acagcggaca agagtagctc aaccgcatac 240
atgcaactgt caagccttac ctccgaagac agcgcagtgt actactgcgc cagaagcacc 300
tactatgggg gtgattggta cttcaacgtc tggggggctg gcaccacagt gactgtaagc 360
gca 363
<210> 92
<211> 106
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 利妥昔单抗 VL
<400> 92
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 93
<211> 318
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 利妥昔单抗 VL
<400> 93
cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60
atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120
agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180
ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240
gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300
actaagttgg aaattaag 318
<210> 94
<211> 450
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 曲妥珠单抗 重链
<400> 94
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 95
<211> 1350
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 曲妥珠单抗 重链
<400> 95
gaagtccaat tggtcgaatc aggcggtgga ctcgtgcaac ctggaggttc gttacgctta 60
tcatgtgctg caagtggatt taatattaaa gatacctaca tccactgggt acgtcaagct 120
cccggcaagg gtctcgagtg ggtcgcacgc atttacccca ccaacggata cacgcgctac 180
gccgattcag tgaagggacg tttcacaatc tctgctgata ctagcaaaaa taccgcatac 240
ctccagatga actctcttag ggccgaggac acagctgtgt actactgtag ccgttgggga 300
ggagacggtt tttacgcaat ggattactgg ggccaaggaa ccctggtcac agtttcatcg 360
gcgtcgacca agggcccgtc agtgttcccg ctggccccgt catccaagtc cacgtctggg 420
ggcacagcag ccctgggatg cttggtcaag gactacttcc ccgagcccgt gactgtgtcc 480
tggaactccg gagcactgac ctccggagtg cacacctttc ccgcggtgct gcagtcctcc 540
ggactgtact ccctgtcgtc ggtcgtgacc gtgccgagct cctcgctcgg aacccagacc 600
tacatctgca acgtgaacca caagccctcg aacaccaaag tggacaagaa ggtcgagccc 660
aaaagctgcg acaagactca cacttgtccg ccgtgccccg cccccgaact gctgggtggc 720
ccctccgtgt tcctgttccc gcctaagcct aaggacaccc ttatgatcag ccgcacccct 780
gaagtgacct gtgtcgtcgt ggatgtgtca cacgaggacc cggaggtcaa gttcaattgg 840
tacgtggacg gcgtggaagt gcataacgca aagaccaagc ctcgggagga acagtacaac 900
tcgacctacc gcgtggtgtc agtcctgact gtgctgcacc aggactggct gaacgggaag 960
gagtacaagt gcaaagtgtc gaacaaggcc ctgccggctc caattgaaaa gaccatcagc 1020
aaggccaagg gccagccaag ggaaccacag gtgtacaccc tccctccttc ccgggacgag 1080
ctgaccaaaa accaagtgtc cctgacttgc cttgtgaagg ggttctaccc ttctgacatt 1140
gccgtcgaat gggaatcgaa cggacagcct gaaaacaact ataagactac cccgcccgtg 1200
ctggattccg acggaagctt cttcctgtac tccaagctga ccgtggacaa gtcgagatgg 1260
cagcagggaa atgtgttcag ctgctccgtg atgcatgagg cgctgcacaa ccactacacc 1320
cagaagtcac tgagcctctc ccccggaaag 1350
<210> 96
<211> 213
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 曲妥珠单抗 轻链
<400> 96
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 97
<211> 642
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 曲妥珠单抗 轻链
<400> 97
gatatccaaa tgactcaatc accatcttca ctctccgcga gcgtgggtga tcgggtcacc 60
atcacatgta gggcgagcca agatgtgaat accgccgtcg cgtggtatca acaaaagccg 120
ggaaaagcac caaaactgct tatatactct gcatccttcc tgtactctgg ggtgccaagc 180
cggttctccg gtagtagatc tggtactgac tttacactca ctatcagcag tctgcaacct 240
gaggactttg cgacatacta ttgccagcag cactacacaa ccccacctac atttggtcag 300
gggacaaagg tggagatcaa gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360
gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420
cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480
tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540
agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccagggactg 600
tccagccccg tgaccaagag cttcaacaga ggagaatgct ag 642
<210> 98
<211> 451
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 利妥昔单抗 重链
<400> 98
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110
Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 99
<211> 1353
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 利妥昔单抗 重链
<400> 99
caagttcagc ttcagcagcc gggggctgag ttggtgaaac ccggggccag tgtgaagatg 60
agctgtaaag cgagcggcta caccttcact tcttataata tgcattgggt taagcaaacg 120
ccaggaaggg ggctggagtg gatcggcgct atttacccag gtaacggtga cacatcatat 180
aaccaaaagt ttaagggaaa ggcaaccctc acagcggaca agagtagctc aaccgcatac 240
atgcaactgt caagccttac ctccgaagac agcgcagtgt actactgcgc cagaagcacc 300
tactatgggg gtgattggta cttcaacgtc tggggggctg gcaccacagt gactgtaagc 360
gcagcgtcga ccaagggccc gtcagtgttc ccgctggccc cgtcatccaa gtccacgtct 420
gggggcacag cagccctggg atgcttggtc aaggactact tccccgagcc cgtgactgtg 480
tcctggaact ccggagcact gacctccgga gtgcacacct ttcccgcggt gctgcagtcc 540
tccggactgt actccctgtc gtcggtcgtg accgtgccga gctcctcgct cggaacccag 600
acctacatct gcaacgtgaa ccacaagccc tcgaacacca aagtggacaa gaaggtcgag 660
cccaaaagct gcgacaagac tcacacttgt ccgccgtgcc ccgcccccga actgctgggt 720
ggcccctccg tgttcctgtt cccgcctaag cctaaggaca cccttatgat cagccgcacc 780
cctgaagtga cctgtgtcgt cgtggatgtg tcacacgagg acccggaggt caagttcaat 840
tggtacgtgg acggcgtgga agtgcataac gcaaagacca agcctcggga ggaacagtac 900
aactcgacct accgcgtggt gtcagtcctg actgtgctgc accaggactg gctgaacggg 960
aaggagtaca agtgcaaagt gtcgaacaag gccctgccgg ctccaattga aaagaccatc 1020
agcaaggcca agggccagcc aagggaacca caggtgtaca ccctccctcc ttcccgggac 1080
gagctgacca aaaaccaagt gtccctgact tgccttgtga aggggttcta cccttctgac 1140
attgccgtcg aatgggaatc gaacggacag cctgaaaaca actataagac taccccgccc 1200
gtgctggatt ccgacggaag cttcttcctg tactccaagc tgaccgtgga caagtcgaga 1260
tggcagcagg gaaatgtgtt cagctgctcc gtgatgcatg aggcgctgca caaccactac 1320
acccagaagt cactgagcct ctcccccgga aag 1353
<210> 100
<211> 212
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 利妥昔单抗 轻链
<400> 100
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Thr Val Ala Ala Pro Ser
100 105 110
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala
115 120 125
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val
130 135 140
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
145 150 155 160
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
165 170 175
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys
180 185 190
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
195 200 205
Arg Gly Glu Cys
210
<210> 101
<211> 639
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 利妥昔单抗 轻链
<400> 101
cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60
atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120
agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180
ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240
gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300
actaagttgg aaattaagac cgtggccgcc ccgagcgtgt tcattttccc tccctccgac 360
gagcagttga aatcgggcac cgctagcgtg gtctgccttc tcaacaattt ctatccacgg 420
gaagccaaag tgcagtggaa ggtcgacaac gcgctccaat ccgggaactc acaggaatcc 480
gtgactgagc aggattccaa ggactcgacc tactccctgt catccacgct gaccctgagc 540
aaggcagact acgagaagca caaggtctac gcctgcgaag tgacacacca gggactgtcc 600
agccccgtga ccaagagctt caacagagga gaatgctag 639
<210> 102
<211> 634
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 曲妥珠单抗 HC_MICwed
<400> 102
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Gly Gly Gly Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val
450 455 460
Leu Ser Trp Asp Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His
465 470 475 480
Leu Asp Gly Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala
485 490 495
Lys Pro Gln Gly Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp
500 505 510
Asp Arg Glu Thr Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met
515 520 525
Thr Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln
530 535 540
Glu Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser
545 550 555 560
Gln His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu
565 570 575
Thr Lys Glu Trp Thr Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala
580 585 590
Met Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr
595 600 605
His Tyr His Ala Met His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr
610 615 620
Leu Lys Ser Gly Val Val Leu Arg Arg Thr
625 630
<210> 103
<211> 1902
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 曲妥珠单抗 HC_MICwed
<400> 103
gaagtccaat tggtcgaatc aggcggtgga ctcgtgcaac ctggaggttc gttacgctta 60
tcatgtgctg caagtggatt taatattaaa gatacctaca tccactgggt acgtcaagct 120
cccggcaagg gtctcgagtg ggtcgcacgc atttacccca ccaacggata cacgcgctac 180
gccgattcag tgaagggacg tttcacaatc tctgctgata ctagcaaaaa taccgcatac 240
ctccagatga actctcttag ggccgaggac acagctgtgt actactgtag ccgttgggga 300
ggagacggtt tttacgcaat ggattactgg ggccaaggaa ccctggtcac agtttcatcg 360
gcgtcgacca agggcccgtc agtgttcccg ctggccccgt catccaagtc cacgtctggg 420
ggcacagcag ccctgggatg cttggtcaag gactacttcc ccgagcccgt gactgtgtcc 480
tggaactccg gagcactgac ctccggagtg cacacctttc ccgcggtgct gcagtcctcc 540
ggactgtact ccctgtcgtc ggtcgtgacc gtgccgagct cctcgctcgg aacccagacc 600
tacatctgca acgtgaacca caagccctcg aacaccaaag tggacaagaa ggtcgagccc 660
aaaagctgcg acaagactca cacttgtccg ccgtgccccg cccccgaact gctgggtggc 720
ccctccgtgt tcctgttccc gcctaagcct aaggacaccc ttatgatcag ccgcacccct 780
gaagtgacct gtgtcgtcgt ggcagtgtca cacgaggacc cggaggtcaa gttcaattgg 840
tacgtggacg gcgtggaagt gcataacgca aagaccaagc ctcgggagga acagtacgcc 900
tcgacctacc gcgtggtgtc agtcctgact gtgctgcacc aggactggct gaacgggaag 960
gagtacaagt gcaaagtgtc gaacaaggcc ctgccggctc caattgaaaa gaccatcagc 1020
aaggccaagg gccagccaag ggaaccacag gtgtacaccc tccctccttc ccgggacgag 1080
ctgaccaaaa accaagtgtc cctgacttgc cttgtgaagg ggttctaccc ttctgacatt 1140
gccgtcgaat gggaatcgaa cggacagcct gaaaacaact ataagactac cccgcccgtg 1200
ctggattccg acggaagctt cttcctgtac tccaagctga ccgtggacaa gtcgagatgg 1260
cagcagggaa atgtgttcag ctgctccgtg atgcatgagg cgctgcacaa ccactacacc 1320
cagaagtcac tgagcctctc ccccggagga ggtggcagcg agcctcacag cctccggtat 1380
aatttgactg tactctcttg ggatggctcc gtgcagtccg gctttctgac tgaagttcat 1440
ctcgacggtc aacctttcct gcgctgcgac cgacaaaaat gccgcgccaa gccccaaggg 1500
cagtgggccg aagatgtact gggaaacaag acctgggacc gggagacacg agacctgaca 1560
ggctggggca aggacttgcg catgacactc gcccatatca aggaccagaa ggaaggattg 1620
cactctttgc aagagattcg cgtgtgtgaa atccacgagg acaattcaac gaggagctcc 1680
cagcacttct attacgatgg agaactcttc ttgtcacaga acttggaaac caaggaatgg 1740
actatgcctc agagctctcg ggcacagact ctcgctatga acgttagaaa cttccttaag 1800
gaggatgcta tggagaccga tactcactac cacgccatgc acgccgactg cctccaggaa 1860
ctgcggagat atctgaagtc cggcgtggtt ttgagaagaa cc 1902
<210> 104
<211> 634
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 曲妥珠单抗 HC_MIC25
<400> 104
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Gly Gly Gly Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val
450 455 460
Leu Ser Trp Asp Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His
465 470 475 480
Leu Asp Gly Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala
485 490 495
Lys Pro Gln Gly Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp
500 505 510
Asp Arg Glu Thr Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met
515 520 525
Thr Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln
530 535 540
Glu Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser
545 550 555 560
Gln His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu
565 570 575
Thr Leu Glu Trp Thr Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala
580 585 590
Met Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr
595 600 605
His Tyr His Ala Met Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr
610 615 620
Leu Lys Ser Gly Val Val Leu Arg Arg Thr
625 630
<210> 105
<211> 1902
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 曲妥珠单抗 HC_MIC25
<400> 105
gaagtccaat tggtcgaatc aggcggtgga ctcgtgcaac ctggaggttc gttacgctta 60
tcatgtgctg caagtggatt taatattaaa gatacctaca tccactgggt acgtcaagct 120
cccggcaagg gtctcgagtg ggtcgcacgc atttacccca ccaacggata cacgcgctac 180
gccgattcag tgaagggacg tttcacaatc tctgctgata ctagcaaaaa taccgcatac 240
ctccagatga actctcttag ggccgaggac acagctgtgt actactgtag ccgttgggga 300
ggagacggtt tttacgcaat ggattactgg ggccaaggaa ccctggtcac agtttcatcg 360
gcgtcgacca agggcccgtc agtgttcccg ctggccccgt catccaagtc cacgtctggg 420
ggcacagcag ccctgggatg cttggtcaag gactacttcc ccgagcccgt gactgtgtcc 480
tggaactccg gagcactgac ctccggagtg cacacctttc ccgcggtgct gcagtcctcc 540
ggactgtact ccctgtcgtc ggtcgtgacc gtgccgagct cctcgctcgg aacccagacc 600
tacatctgca acgtgaacca caagccctcg aacaccaaag tggacaagaa ggtcgagccc 660
aaaagctgcg acaagactca cacttgtccg ccgtgccccg cccccgaact gctgggtggc 720
ccctccgtgt tcctgttccc gcctaagcct aaggacaccc ttatgatcag ccgcacccct 780
gaagtgacct gtgtcgtcgt ggcagtgtca cacgaggacc cggaggtcaa gttcaattgg 840
tacgtggacg gcgtggaagt gcataacgca aagaccaagc ctcgggagga acagtacgcc 900
tcgacctacc gcgtggtgtc agtcctgact gtgctgcacc aggactggct gaacgggaag 960
gagtacaagt gcaaagtgtc gaacaaggcc ctgccggctc caattgaaaa gaccatcagc 1020
aaggccaagg gccagccaag ggaaccacag gtgtacaccc tccctccttc ccgggacgag 1080
ctgaccaaaa accaagtgtc cctgacttgc cttgtgaagg ggttctaccc ttctgacatt 1140
gccgtcgaat gggaatcgaa cggacagcct gaaaacaact ataagactac cccgcccgtg 1200
ctggattccg acggaagctt cttcctgtac tccaagctga ccgtggacaa gtcgagatgg 1260
cagcagggaa atgtgttcag ctgctccgtg atgcatgagg cgctgcacaa ccactacacc 1320
cagaagtcac tgagcctctc ccccggagga ggtggcagcg agcctcacag cctccggtat 1380
aatttgactg tactctcttg ggatggctcc gtgcagtccg gctttctgac tgaagttcat 1440
ctcgacggtc aacctttcct gcgctgcgac cgacaaaaat gccgcgccaa gccccaaggg 1500
cagtgggccg aagatgtact gggaaacaag acctgggacc gggagacacg agacctgaca 1560
ggctggggca aggacttgcg catgacactc gcccatatca aggaccagaa ggaaggattg 1620
cactctttgc aagagattcg cgtgtgtgaa atccacgagg acaattcaac gaggagctcc 1680
cagcacttct attacgatgg agaactcttc ttgtcacaga acttggaaac cctcgaatgg 1740
actatgcctc agagctctcg ggcacagact ctcgctatga acgttagaaa cttccttaag 1800
gaggatgcta tggagaccga tactcactac cacgccatgc gcgccgactg cctctctgaa 1860
ctgcggagat atctgaagtc cggcgtggtt ttgagaagaa cc 1902
<210> 106
<211> 404
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 利妥昔单抗 LC_ULBP2.wt
<400> 106
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly Gly Gly Ser
210 215 220
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
225 230 235 240
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
245 250 255
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
260 265 270
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
275 280 285
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Arg Asp Ile
290 295 300
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
305 310 315 320
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
325 330 335
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
340 345 350
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
355 360 365
Glu Asn Asp Lys Val Val Ala Met Ser Phe His Tyr Phe Ser Met Gly
370 375 380
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
385 390 395 400
Leu Glu Pro Ser
<210> 107
<211> 1212
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 利妥昔单抗 LC_ULBP2.wt
<400> 107
cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60
atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120
agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180
ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240
gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300
actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360
gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420
cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480
tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540
agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccagggactg 600
tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660
ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720
cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780
gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840
acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900
ttgcgcgaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960
atgtcttgcg agcaaaaggc agagggccac tcctccggca gctggcagtt cagtttcgac 1020
ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080
gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgatgtc attccactat 1140
ttctcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200
ttggagccat cg 1212
<210> 108
<211> 187
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 ULPB2 α1-α2 变体 R80W
<400> 108
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
1 5 10 15
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
20 25 30
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
35 40 45
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
50 55 60
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile
65 70 75 80
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
85 90 95
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
100 105 110
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
115 120 125
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
130 135 140
Glu Asn Asp Lys Val Val Ala Met Ser Phe His Tyr Phe Ser Met Gly
145 150 155 160
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
165 170 175
Leu Glu Pro Ser Ala Gly Ala Pro Pro Met Val
180 185
<210> 109
<211> 540
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULPB2 α1-α2 变体 R80W
<400> 109
gagccccata gtctgagcta cgacatcaca gttattccca agttcaggcc cggaccgcgc 60
tggtgtgccg tgcaaggaca agtcgacgaa aaaacctttc ttcattacga ttgcggaaat 120
aagactgtaa cgccagtctc tcctttaggt aagaagttaa acgtcactac ggcgtggaag 180
gcacaaaacc ccgtcctgcg cgaggtcgtc gacatcctga ctgaacaatt gtgggacatc 240
cagctcgaga attacactcc aaaggagcct cttaccctgc aggctagaat gtcttgcgag 300
caaaaggcag agggccactc ctccggcagc tggcagttca gtttcgacgg acaaatcttt 360
ctgttattcg attcagagaa gagaatgtgg actacagttc accccggtgc ccgtaaaatg 420
aaggagaagt gggaaaacga caaagtggtg gcgatgtcat tccactattt ctcgatggga 480
gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540
<210> 110
<211> 513
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULPB2 α1-α2 变体 R80W NNK 文库
<220>
<221> misc_feature
<222> (457)..(458)
<223> n 是 a、c、g、或 t
<220>
<221> misc_feature
<222> (460)..(461)
<223> n 是 a、c、g、或 t
<220>
<221> misc_feature
<222> (463)..(464)
<223> n 是 a、c、g、或 t
<220>
<221> misc_feature
<222> (466)..(467)
<223> n 是 a、c、g、或 t
<220>
<221> misc_feature
<222> (469)..(470)
<223> n 是 a、c、g、或 t
<400> 110
gagccccata gcctcagcta tgacattacg gtgattccca aatttcgccc aggaccacgt 60
tggtgcgccg tccagggtca ggtagatgaa aagactttcc tgcattatga ttgcggcaat 120
aaaaccgtga cgccggtatc gccgttaggc aaaaaattga atgtcacgac agcgtggaaa 180
gcacagaacc cggtgttgcg cgaggtagtc gatattttga cggaacaact ctgggacatt 240
cagctcgaga attacacccc aaaagaaccg ctgacgctgc aagcgcgtat gtcgtgcgaa 300
caaaaagcag aaggtcactc tagcgggagt tggcagtttt ccttcgatgg gcagattttt 360
ctgctgtttg attcggagaa acgcatgtgg actacagtcc acccgggtgc ccggaaaatg 420
aaagagaagt gggagaatga taaagtggtg gccactnnkn nknnknnknn ktccatgggc 480
gattgcattg gctggttaga ggattttctc atg 513
<210> 111
<211> 180
<212> PRT
<213> 人工序列
<220>
<223> 合成肽, ULBP2 α1-α2 变体 ULBP2.C
<400> 111
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
1 5 10 15
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
20 25 30
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
35 40 45
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
50 55 60
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile
65 70 75 80
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
85 90 95
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
100 105 110
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
115 120 125
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
130 135 140
Glu Asn Asp Lys Val Val Ala Thr Ile Leu Trp Gln Thr Ser Met Gly
145 150 155 160
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
165 170 175
Leu Glu Pro Ser
180
<210> 112
<211> 540
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULBP2 α1-α2 变体 ULBP2.C
<400> 112
gagccccata gtctgagcta cgacatcaca gttattccca agttcaggcc cggaccgcgc 60
tggtgtgccg tgcaaggaca agtcgacgaa aaaacctttc ttcattacga ttgcggaaat 120
aagactgtaa cgccagtctc tcctttaggt aagaagttaa acgtcactac ggcgtggaag 180
gcacaaaacc ccgtcctgcg cgaggtcgtc gacatcctga ctgaacaatt gtgggacatc 240
cagctcgaga attacactcc aaaggagcct cttaccctgc aggctagaat gtcttgcgag 300
caaaaggcag agggccactc ctccggcagc tggcagttca gtttcgacgg acaaatcttt 360
ctgttattcg attcagagaa gagaatgtgg actacagttc accccggtgc ccgtaaaatg 420
aaggagaagt gggaaaacga caaagtggtg gcgactattc tgtggcagac ttcgatggga 480
gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540
<210> 113
<211> 180
<212> PRT
<213> 人工序列
<220>
<223> 合成肽, ULBP2 α1-α2 变体 ULBP2.R
<400> 113
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
1 5 10 15
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
20 25 30
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
35 40 45
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
50 55 60
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile
65 70 75 80
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
85 90 95
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
100 105 110
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
115 120 125
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
130 135 140
Glu Asn Asp Lys Val Val Ala Thr Leu Leu Trp Gly Trp Ser Met Gly
145 150 155 160
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
165 170 175
Leu Glu Pro Ser
180
<210> 114
<211> 540
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULBP2 α1-α2 变体 ULBP2.R
<400> 114
gagccccata gtctgagcta cgacatcaca gttattccca agttcaggcc cggaccgcgc 60
tggtgtgccg tgcaaggaca agtcgacgaa aaaacctttc ttcattacga ttgcggaaat 120
aagactgtaa cgccagtctc tcctttaggt aagaagttaa acgtcactac ggcgtggaag 180
gcacaaaacc ccgtcctgcg cgaggtcgtc gacatcctga ctgaacaatt gtgggacatc 240
cagctcgaga attacactcc aaaggagcct cttaccctgc aggctagaat gtcttgcgag 300
caaaaggcag agggccactc ctccggcagc tggcagttca gtttcgacgg acaaatcttt 360
ctgttattcg attcagagaa gagaatgtgg actacagttc accccggtgc ccgtaaaatg 420
aaggagaagt gggaaaacga caaagtggtg gcgactttgt tgtgggggtg gtcgatggga 480
gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540
<210> 115
<211> 180
<212> PRT
<213> 人工序列
<220>
<223> 合成肽, ULBP2 α1-α2 变体 ULBP2.AA
<400> 115
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
1 5 10 15
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
20 25 30
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
35 40 45
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
50 55 60
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile
65 70 75 80
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
85 90 95
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
100 105 110
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
115 120 125
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
130 135 140
Glu Asn Asp Lys Val Val Ala Thr Met Phe Trp Ser Trp Ser Met Gly
145 150 155 160
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
165 170 175
Leu Glu Pro Ser
180
<210> 116
<211> 540
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULBP2 α1-α2 变体 ULBP2.AA
<400> 116
gagccccata gtctgagcta cgacatcaca gttattccca agttcaggcc cggaccgcgc 60
tggtgtgccg tgcaaggaca agtcgacgaa aaaacctttc ttcattacga ttgcggaaat 120
aagactgtaa cgccagtctc tcctttaggt aagaagttaa acgtcactac ggcgtggaag 180
gcacaaaacc ccgtcctgcg cgaggtcgtc gacatcctga ctgaacaatt gtgggacatc 240
cagctcgaga attacactcc aaaggagcct cttaccctgc aggctagaat gtcttgcgag 300
caaaaggcag agggccactc ctccggcagc tggcagttca gtttcgacgg acaaatcttt 360
ctgttattcg attcagagaa gagaatgtgg actacagttc accccggtgc ccgtaaaatg 420
aaggagaagt gggaaaacga caaagtggtg gcgactatgt tttggagttg gtcgatggga 480
gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540
<210> 117
<211> 180
<212> PRT
<213> 人工序列
<220>
<223> 合成肽, ULBP2 α1-α2 变体 ULBP2.AB
<400> 117
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
1 5 10 15
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
20 25 30
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
35 40 45
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
50 55 60
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile
65 70 75 80
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
85 90 95
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
100 105 110
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
115 120 125
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
130 135 140
Glu Asn Asp Lys Val Val Ala Thr Leu Met Trp Gln Trp Ser Met Gly
145 150 155 160
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
165 170 175
Leu Glu Pro Ser
180
<210> 118
<211> 540
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULBP2 α1-α2 变体 ULBP2.AB
<400> 118
gagccccata gtctgagcta cgacatcaca gttattccca agttcaggcc cggaccgcgc 60
tggtgtgccg tgcaaggaca agtcgacgaa aaaacctttc ttcattacga ttgcggaaat 120
aagactgtaa cgccagtctc tcctttaggt aagaagttaa acgtcactac ggcgtggaag 180
gcacaaaacc ccgtcctgcg cgaggtcgtc gacatcctga ctgaacaatt gtgggacatc 240
cagctcgaga attacactcc aaaggagcct cttaccctgc aggctagaat gtcttgcgag 300
caaaaggcag agggccactc ctccggcagc tggcagttca gtttcgacgg acaaatcttt 360
ctgttattcg attcagagaa gagaatgtgg actacagttc accccggtgc ccgtaaaatg 420
aaggagaagt gggaaaacga caaagtggtg gcgactctta tgtggcagtg gtcgatggga 480
gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540
<210> 119
<211> 404
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 利妥昔单抗 LC_ULBP2.C
<400> 119
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly Gly Gly Ser
210 215 220
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
225 230 235 240
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
245 250 255
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
260 265 270
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
275 280 285
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile
290 295 300
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
305 310 315 320
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
325 330 335
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
340 345 350
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
355 360 365
Glu Asn Asp Lys Val Val Ala Thr Ile Leu Trp Gln Thr Ser Met Gly
370 375 380
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
385 390 395 400
Leu Glu Pro Ser
<210> 120
<211> 1212
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 利妥昔单抗 LC_ULBP2.C
<400> 120
cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60
atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120
agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180
ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240
gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300
actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360
gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420
cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480
tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540
agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccagggactg 600
tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660
ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720
cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780
gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840
acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900
ttgtgggaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960
atgtcttgcg agcaaaaggc agagggccac tcctccggca gctggcagtt cagtttcgac 1020
ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080
gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgactat tctgtggcag 1140
acttcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200
ttggagccat cg 1212
<210> 121
<211> 404
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 利妥昔单抗 LC_ULBP2.R
<400> 121
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly Gly Gly Ser
210 215 220
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
225 230 235 240
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
245 250 255
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
260 265 270
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
275 280 285
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile
290 295 300
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
305 310 315 320
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
325 330 335
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
340 345 350
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
355 360 365
Glu Asn Asp Lys Val Val Ala Thr Leu Leu Trp Gly Trp Ser Met Gly
370 375 380
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
385 390 395 400
Leu Glu Pro Ser
<210> 122
<211> 1212
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 利妥昔单抗 LC_ULBP2.R
<400> 122
cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60
atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120
agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180
ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240
gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300
actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360
gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420
cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480
tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540
agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccagggactg 600
tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660
ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720
cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780
gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840
acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900
ttgtgggaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960
atgtcttgcg agcaaaaggc agagggccac tcctccggca gctggcagtt cagtttcgac 1020
ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080
gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgacttt gttgtggggg 1140
tggtcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200
ttggagccat cg 1212
<210> 123
<211> 404
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 利妥昔单抗 LC_ULBP2.AA
<400> 123
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly Gly Gly Ser
210 215 220
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
225 230 235 240
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
245 250 255
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
260 265 270
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
275 280 285
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile
290 295 300
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
305 310 315 320
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
325 330 335
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
340 345 350
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
355 360 365
Glu Asn Asp Lys Val Val Ala Thr Met Phe Trp Ser Trp Ser Met Gly
370 375 380
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
385 390 395 400
Leu Glu Pro Ser
<210> 124
<211> 1212
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 利妥昔单抗 LC_ULBP2.AA
<400> 124
cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60
atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120
agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180
ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240
gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300
actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360
gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420
cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480
tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540
agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccagggactg 600
tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660
ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720
cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780
gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840
acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900
ttgtgggaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960
atgtcttgcg agcaaaaggc agagggccac tcctccggca gctggcagtt cagtttcgac 1020
ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080
gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgactat gttttggagt 1140
tggtcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200
ttggagccat cg 1212
<210> 125
<211> 404
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 利妥昔单抗 LC_ULBP2.AB
<400> 125
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly Gly Gly Ser
210 215 220
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
225 230 235 240
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
245 250 255
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
260 265 270
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
275 280 285
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile
290 295 300
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
305 310 315 320
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
325 330 335
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
340 345 350
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
355 360 365
Glu Asn Asp Lys Val Val Ala Thr Leu Met Trp Gln Trp Ser Met Gly
370 375 380
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
385 390 395 400
Leu Glu Pro Ser
<210> 126
<211> 1212
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 利妥昔单抗 LC_ULBP2.AB
<400> 126
cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60
atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120
agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180
ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240
gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300
actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360
gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420
cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480
tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540
agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccagggactg 600
tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660
ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720
cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780
gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840
acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900
ttgtgggaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960
atgtcttgcg agcaaaaggc agagggccac tcctccggca gctggcagtt cagtttcgac 1020
ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080
gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgactct tatgtggcag 1140
tggtcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200
ttggagccat cg 1212
<210> 127
<211> 180
<212> PRT
<213> 人工序列
<220>
<223> 合成肽, ULBP2 α1-α2 变体 ULBP2.S3
<400> 127
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
1 5 10 15
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
20 25 30
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
35 40 45
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
50 55 60
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile
65 70 75 80
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
85 90 95
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
100 105 110
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
115 120 125
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
130 135 140
Glu Asn Asp Lys Val Val Ala Thr Lys Leu Tyr Leu Trp Ser Met Gly
145 150 155 160
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
165 170 175
Leu Glu Pro Ser
180
<210> 128
<211> 540
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 ULBP2 α1-α2 变体 ULBP2.S3
<400> 128
gagccccata gtctgagcta cgacatcaca gttattccca agttcaggcc cggaccgcgc 60
tggtgtgccg tgcaaggaca agtcgacgaa aaaacctttc ttcattacga ttgcggaaat 120
aagactgtaa cgccagtctc tcctttaggt aagaagttaa acgtcactac ggcgtggaag 180
gcacaaaacc ccgtcctgcg cgaggtcgtc gacatcctga ctgaacaatt gtgggacatc 240
cagctcgaga attacactcc aaaggagcct cttaccctgc aggctagaat gtcttgcgag 300
caaaaggcag agggccactc ctccggcagc tggcagttca gtttcgacgg acaaatcttt 360
ctgttattcg attcagagaa gagaatgtgg actacagttc accccggtgc ccgtaaaatg 420
aaggagaagt gggaaaacga caaagtggtg gcgactaagc tttatctttg gtcgatggga 480
gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540
<210> 129
<211> 404
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 利妥昔单抗 LC_ULBP2.S3
<400> 129
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly Gly Gly Ser
210 215 220
Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg
225 230 235 240
Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr
245 250 255
Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro
260 265 270
Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro
275 280 285
Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile
290 295 300
Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg
305 310 315 320
Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln
325 330 335
Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg
340 345 350
Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp
355 360 365
Glu Asn Asp Lys Val Val Ala Thr Lys Leu Tyr Leu Trp Ser Met Gly
370 375 380
Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr
385 390 395 400
Leu Glu Pro Ser
<210> 130
<211> 1212
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 利妥昔单抗 LC_ULBP2.S3
<400> 130
cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60
atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120
agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180
ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240
gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300
actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360
gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420
cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480
tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540
agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccagggactg 600
tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660
ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720
cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780
gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840
acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900
ttgtgggaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960
atgtcttgcg agcaaaaggc agagggccac tcctccggca gctggcagtt cagtttcgac 1020
ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080
gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgactaa gctttatctt 1140
tggtcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200
ttggagccat cg 1212
<210> 131
<211> 634
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 利妥昔单抗 HC_ULBP2.R80W
<400> 131
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110
Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Gly Gly Gly Ser Glu Pro His Ser Leu Ser Tyr Asp Ile Thr
450 455 460
Val Ile Pro Lys Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly
465 470 475 480
Gln Val Asp Glu Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr
485 490 495
Val Thr Pro Val Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala
500 505 510
Trp Lys Ala Gln Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr
515 520 525
Glu Gln Leu Trp Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro
530 535 540
Leu Thr Leu Gln Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His
545 550 555 560
Ser Ser Gly Ser Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu
565 570 575
Phe Asp Ser Glu Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg
580 585 590
Lys Met Lys Glu Lys Trp Glu Asn Asp Lys Val Val Ala Met Ser Phe
595 600 605
His Tyr Phe Ser Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu
610 615 620
Met Gly Met Asp Ser Thr Leu Glu Pro Ser
625 630
<210> 132
<211> 1902
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 利妥昔单抗 HC_ULBP2.R80W
<400> 132
caagttcagc ttcagcagcc gggggctgag ttggtgaaac ccggggccag tgtgaagatg 60
agctgtaaag cgagcggcta caccttcact tcttataata tgcattgggt taagcaaacg 120
ccaggaaggg ggctggagtg gatcggcgct atttacccag gtaacggtga cacatcatat 180
aaccaaaagt ttaagggaaa ggcaaccctc acagcggaca agagtagctc aaccgcatac 240
atgcaactgt caagccttac ctccgaagac agcgcagtgt actactgcgc cagaagcacc 300
tactatgggg gtgattggta cttcaacgtc tggggggctg gcaccacagt gactgtaagc 360
gcagcgtcga ccaagggccc gtcagtgttc ccgctggccc cgtcatccaa gtccacgtct 420
gggggcacag cagccctggg atgcttggtc aaggactact tccccgagcc cgtgactgtg 480
tcctggaact ccggagcact gacctccgga gtgcacacct ttcccgcggt gctgcagtcc 540
tccggactgt actccctgtc gtcggtcgtg accgtgccga gctcctcgct cggaacccag 600
acctacatct gcaacgtgaa ccacaagccc tcgaacacca aagtggacaa gaaggtcgag 660
cccaaaagct gcgacaagac tcacacttgt ccgccgtgcc ccgcccccga actgctgggt 720
ggcccctccg tgttcctgtt cccgcctaag cctaaggaca cccttatgat cagccgcacc 780
cctgaagtga cctgtgtcgt cgtggcagtg tcacacgagg acccggaggt caagttcaat 840
tggtacgtgg acggcgtgga agtgcataac gcaaagacca agcctcggga ggaacagtac 900
gcctcgacct accgcgtggt gtcagtcctg actgtgctgc accaggactg gctgaacggg 960
aaggagtaca agtgcaaagt gtcgaacaag gccctgccgg ctccaattga aaagaccatc 1020
agcaaggcca agggccagcc aagggaacca caggtgtaca ccctccctcc ttcccgggac 1080
gagctgacca aaaaccaagt gtccctgact tgccttgtga aggggttcta cccttctgac 1140
attgccgtcg aatgggaatc gaacggacag cctgaaaaca actataagac taccccgccc 1200
gtgctggatt ccgacggaag cttcttcctg tactccaagc tgaccgtgga caagtcgaga 1260
tggcagcagg gaaatgtgtt cagctgctcc gtgatgcatg aggcgctgca caaccactac 1320
acccagaagt cactgagcct ctcccccgga ggaggtggca gcgagcccca tagtctgagc 1380
tacgacatca cagttattcc caagttcagg cccggaccgc gctggtgtgc cgtgcaagga 1440
caagtcgacg aaaaaacctt tcttcattac gattgcggaa ataagactgt aacgccagtc 1500
tctcctttag gtaagaagtt aaacgtcact acggcgtgga aggcacaaaa ccccgtcctg 1560
cgcgaggtcg tcgacatcct gactgaacaa ttgtgggaca tccagctcga gaattacact 1620
ccaaaggagc ctcttaccct gcaggctaga atgtcttgcg agcaaaaggc agagggccac 1680
tcctccggca gctggcagtt cagtttcgac ggacaaatct ttctgttatt cgattcagag 1740
aagagaatgt ggactacagt tcaccccggt gcccgtaaaa tgaaggagaa gtgggaaaac 1800
gacaaagtgg tggcgatgtc attccactat ttctcgatgg gagactgcat cggttggctg 1860
gaagatttcc tcatgggtat ggactccact ttggagccat cg 1902
<210> 133
<211> 405
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 曲妥珠单抗 LC_ULBP2.R
<400> 133
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly Gly Gly
210 215 220
Ser Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe
225 230 235 240
Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys
245 250 255
Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser
260 265 270
Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn
275 280 285
Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp
290 295 300
Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala
305 310 315 320
Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp
325 330 335
Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys
340 345 350
Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys
355 360 365
Trp Glu Asn Asp Lys Val Val Ala Thr Leu Leu Trp Gly Trp Ser Met
370 375 380
Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser
385 390 395 400
Thr Leu Glu Pro Ser
405
<210> 134
<211> 1215
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 曲妥珠单抗 LC_ULBP2.R
<400> 134
gatatccaaa tgactcaatc accatcttca ctctccgcga gcgtgggtga tcgggtcacc 60
atcacatgta gggcgagcca agatgtgaat accgccgtcg cgtggtatca acaaaagccg 120
ggaaaagcac caaaactgct tatatactct gcatccttcc tgtactctgg ggtgccaagc 180
cggttctccg gtagtagatc tggtactgac tttacactca ctatcagcag tctgcaacct 240
gaggactttg cgacatacta ttgccagcag cactacacaa ccccacctac atttggtcag 300
gggacaaagg tggagatcaa gcggaccgtg gccgccccga gcgtgttcat tttccctccc 360
tccgacgagc agttgaaatc gggcaccgct agcgtggtct gccttctcaa caatttctat 420
ccacgggaag ccaaagtgca gtggaaggtc gacaacgcgc tccaatccgg gaactcacag 480
gaatccgtga ctgagcagga ttccaaggac tcgacctact ccctgtcatc cacgctgacc 540
ctgagcaagg cagactacga gaagcacaag gtctacgcct gcgaagtgac acaccaggga 600
ctgtccagcc ccgtgaccaa gagcttcaac agaggagaat gcgcacctac ctcaagctct 660
ggaggaggtg gcagcgagcc ccatagtctg agctacgaca tcacagttat tcccaagttc 720
aggcccggac cgcgctggtg tgccgtgcaa ggacaagtcg acgaaaaaac ctttcttcat 780
tacgattgcg gaaataagac tgtaacgcca gtctctcctt taggtaagaa gttaaacgtc 840
actacggcgt ggaaggcaca aaaccccgtc ctgcgcgagg tcgtcgacat cctgactgaa 900
caattgtggg acatccagct cgagaattac actccaaagg agcctcttac cctgcaggct 960
agaatgtctt gcgagcaaaa ggcagagggc cactcctccg gcagctggca gttcagtttc 1020
gacggacaaa tctttctgtt attcgattca gagaagagaa tgtggactac agttcacccc 1080
ggtgcccgta aaatgaagga gaagtgggaa aacgacaaag tggtggcgac tttgttgtgg 1140
gggtggtcga tgggagactg catcggttgg ctggaagatt tcctcatggg tatggactcc 1200
actttggagc catcg 1215
<210> 135
<211> 156
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 NKG2D.wt 胞外域
<400> 135
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
145 150 155
<210> 136
<211> 468
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 NKG2D.wt 胞外域
<400> 136
atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60
cctctgttca atcaagaggt gcagatccct ctgaccgaga gctactgtgg cccctgtcct 120
aagaactgga tctgctacaa gaacaactgc taccagttct tcgacgagag caagaattgg 180
tacgagagcc aggccagctg catgagccag aatgccagcc tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtcaag agctaccact ggatgggact cgtgcacatc 300
cctacaaacg gcagctggca gtgggaggac ggctctatcc tgtctcctaa cctgctgacc 360
atcatcgaga tgcagaaggg cgactgcgcc ctgtacgcca gcagctttaa gggctacatc 420
gagaactgca gcacccctaa cacctacatc tgtatgcagc ggaccgtg 468
<210> 137
<211> 156
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 NKG2D.YA 胞外域
<400> 137
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
145 150 155
<210> 138
<211> 468
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 NKG2D.YA 胞外域
<400> 138
atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60
cctctgttca atcaagaggt gcagatccct ctgaccgaga gctactgtgg cccctgtcct 120
aagaactgga tctgctacaa gaacaactgc taccagttct tcgacgagag caagaattgg 180
tacgagagcc aggccagctg catgagccag aatgccagcc tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtcaag agcgcccact ggatgggact cgtgcacatc 300
cctacaaacg gcagctggca gtgggaggac ggctctatcc tgtctcctaa cctgctgacc 360
atcatcgaga tgcagaaggg cgactgcgcc ctgtacgcca gcagctttaa gggctacatc 420
gagaactgca gcacccctaa cacctacatc tgtatgcagc ggaccgtg 468
<210> 139
<211> 156
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 NKG2D.AF 胞外域
<400> 139
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
145 150 155
<210> 140
<211> 468
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 NKG2D.AF 胞外域
<400> 140
atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60
cctctgttca atcaagaggt gcagatccct ctgaccgaga gctactgtgg cccctgtcct 120
aagaactgga tctgctacaa gaacaactgc taccagttct tcgacgagag caagaattgg 180
tacgagagcc aggccagctg catgagccag aatgccagcc tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtcaag agcgcccact ggatgggact cgtgcacatc 300
cctacaaacg gcagctggca gtgggaggac ggctctatcc tgtctcctaa cctgctgacc 360
atcatcgaga tgcagaaggg cgactgcgcc ctgtacgcca gcagctttaa gggcttcatc 420
gagaactgca gcacccctaa cacctacatc tgtatgcagc ggaccgtg 468
<210> 141
<211> 69
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 CD8α 铰链和跨膜结构域
<400> 141
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
35 40 45
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
50 55 60
Ile Thr Leu Tyr Cys
65
<210> 142
<211> 207
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 CD8α 铰链和跨膜结构域
<400> 142
accaccacac cagctcctag acctccaact cctgctccta caatcgccag ccagcctctg 60
tctctgaggc cagaagcttg tagacctgct gcaggcggag ccgtgcatac aagaggactg 120
gatttcgcct gcgacatcta catctgggcc cctctggctg gaacatgtgg cgtgctgctg 180
ctgagcctgg tcatcaccct gtactgc 207
<210> 143
<211> 42
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 4-1BB
<400> 143
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 144
<211> 126
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 4-1BB
<400> 144
aagcggggca gaaagaagct gctgtacatc tttaagcagc ccttcatgcg gcccgtgcag 60
accacacaag aggaagatgg ctgctcctgc agattccccg aggaagaaga aggcggctgc 120
gagctg 126
<210> 145
<211> 112
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 CD3ζ
<400> 145
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 146
<211> 336
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 CD3ζ
<400> 146
agagtgaagt tcagccgttc tgccgacgct cccgcctata agcagggaca gaaccagctg 60
tacaacgagc tgaacctggg gagaagagaa gagtacgacg tgctggacaa gcggagaggc 120
agagatcctg agatgggcgg caagcccaga cggaagaatc ctcaagaggg cctgtataat 180
gagctgcaga aagacaagat ggccgaggcc tacagcgaga tcggaatgaa gggcgagcgc 240
agaagaggca agggacacga tggactgtac cagggcctga gcaccgccac caaggatacc 300
tatgatgccc tgcacatgca ggccctgcct ccaaga 336
<210> 147
<211> 239
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 EGFP
<400> 147
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 148
<211> 717
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 EGFP
<400> 148
atggtgtcta aaggcgagga actgttcacc ggcgtggtgc ccattctggt ggaactggac 60
ggggatgtga acggccacaa gtttagcgtt agcggcgaag gcgaagggga tgccacatac 120
ggaaagctga ccctgaagtt catctgcacc accggcaagc tgcctgtgcc ttggcctaca 180
ctggtcacca cactgacata cggcgtgcag tgctttagca gataccccga ccatatgaag 240
cagcacgact tcttcaagtc cgccatgcct gagggctacg tgcaagagcg gaccatcttc 300
tttaaggacg acggcaacta caagaccagg gccgaagtga agtttgaggg cgacaccctg 360
gtcaaccgga tcgagctgaa gggcatcgac ttcaaagagg atggcaacat cctgggccac 420
aagctcgagt acaactacaa cagccacaac gtgtacatca tggccgacaa gcagaagaac 480
ggcatcaagg ccaacttcaa gatccggcac aacatcgagg acggcagcgt tcagctggcc 540
gatcactacc agcagaacac ccctatcgga gatggccctg tgctgctccc cgacaatcac 600
tacctgagca cacagagcgc cctgagcaag gaccccaacg agaagaggga tcacatggtg 660
ctgctggaat ttgtgaccgc cgcaggcatc accctcggca tggacgaact gtacaaa 717
<210> 149
<211> 473
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 CD8铰链TM_4-1BB_CD3ζ_EGFP
<400> 149
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
35 40 45
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
50 55 60
Ile Thr Leu Tyr Cys Ser Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr
65 70 75 80
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
85 90 95
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
100 105 110
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln
115 120 125
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
130 135 140
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
145 150 155 160
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
165 170 175
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
180 185 190
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
195 200 205
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
210 215 220
Arg Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Val Ser Lys Gly Glu
225 230 235 240
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp
245 250 255
Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
260 265 270
Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu
275 280 285
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln
290 295 300
Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
305 310 315 320
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys
325 330 335
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
340 345 350
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp
355 360 365
Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn
370 375 380
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
385 390 395 400
Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His
405 410 415
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
420 425 430
Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu
435 440 445
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
450 455 460
Thr Leu Gly Met Asp Glu Leu Tyr Lys
465 470
<210> 150
<211> 1419
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码 CD8铰链TM_4-1BB_CD3ζ_EGFP
<400> 150
accaccacac cagctcctag acctccaact cctgctccta caatcgccag ccagcctctg 60
tctctgaggc cagaagcttg tagacctgct gcaggcggag ccgtgcatac aagaggactg 120
gatttcgcct gcgacatcta catctgggcc cctctggctg gaacatgtgg cgtgctgctg 180
ctgagcctgg tcatcaccct gtactgcagc ctgaagcggg gcagaaagaa gctgctgtac 240
atctttaagc agcccttcat gcggcccgtg cagaccacac aagaggaaga tggctgctcc 300
tgcagattcc ccgaggaaga agaaggcggc tgcgagctga gagtgaagtt cagccgttct 360
gccgacgctc ccgcctataa gcagggacag aaccagctgt acaacgagct gaacctgggg 420
agaagagaag agtacgacgt gctggacaag cggagaggca gagatcctga gatgggcggc 480
aagcccagac ggaagaatcc tcaagagggc ctgtataatg agctgcagaa agacaagatg 540
gccgaggcct acagcgagat cggaatgaag ggcgagcgca gaagaggcaa gggacacgat 600
ggactgtacc agggcctgag caccgccacc aaggatacct atgatgccct gcacatgcag 660
gccctgcctc caagatcagg ctctggttct ggcagcggca gcatggtgtc taaaggcgag 720
gaactgttca ccggcgtggt gcccattctg gtggaactgg acggggatgt gaacggccac 780
aagtttagcg ttagcggcga aggcgaaggg gatgccacat acggaaagct gaccctgaag 840
ttcatctgca ccaccggcaa gctgcctgtg ccttggccta cactggtcac cacactgaca 900
tacggcgtgc agtgctttag cagatacccc gaccatatga agcagcacga cttcttcaag 960
tccgccatgc ctgagggcta cgtgcaagag cggaccatct tctttaagga cgacggcaac 1020
tacaagacca gggccgaagt gaagtttgag ggcgacaccc tggtcaaccg gatcgagctg 1080
aagggcatcg acttcaaaga ggatggcaac atcctgggcc acaagctcga gtacaactac 1140
aacagccaca acgtgtacat catggccgac aagcagaaga acggcatcaa ggccaacttc 1200
aagatccggc acaacatcga ggacggcagc gttcagctgg ccgatcacta ccagcagaac 1260
acccctatcg gagatggccc tgtgctgctc cccgacaatc actacctgag cacacagagc 1320
gccctgagca aggaccccaa cgagaagagg gatcacatgg tgctgctgga atttgtgacc 1380
gccgcaggca tcaccctcgg catggacgaa ctgtacaaa 1419
<210> 151
<211> 629
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 NKG2D.wt_CD8铰链TM_4-1BB_CD3ζ_EGFP 完全嵌合抗原受体
<400> 151
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro
145 150 155 160
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
165 170 175
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
180 185 190
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
195 200 205
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
210 215 220
Cys Ser Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
225 230 235 240
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
245 250 255
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
260 265 270
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
275 280 285
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
290 295 300
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
305 310 315 320
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
325 330 335
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
340 345 350
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
355 360 365
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
370 375 380
Gly Ser Gly Ser Gly Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr
385 390 395 400
Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His
405 410 415
Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys
420 425 430
Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp
435 440 445
Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg
450 455 460
Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro
465 470 475 480
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn
485 490 495
Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn
500 505 510
Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu
515 520 525
Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met
530 535 540
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
545 550 555 560
Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
565 570 575
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
580 585 590
Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
595 600 605
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
610 615 620
Asp Glu Leu Tyr Lys
625
<210> 152
<211> 1887
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码NKG2D.wt_CD8铰链TM_4-1BB_CD3ζ_EGFP 完全嵌合抗原受体
<400> 152
atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60
cctctgttca atcaagaggt gcagatccct ctgaccgaga gctactgtgg cccctgtcct 120
aagaactgga tctgctacaa gaacaactgc taccagttct tcgacgagag caagaattgg 180
tacgagagcc aggccagctg catgagccag aatgccagcc tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtcaag agctaccact ggatgggact cgtgcacatc 300
cctacaaacg gcagctggca gtgggaggac ggctctatcc tgtctcctaa cctgctgacc 360
atcatcgaga tgcagaaggg cgactgcgcc ctgtacgcca gcagctttaa gggctacatc 420
gagaactgca gcacccctaa cacctacatc tgtatgcagc ggaccgtgac caccacacca 480
gctcctagac ctccaactcc tgctcctaca atcgccagcc agcctctgtc tctgaggcca 540
gaagcttgta gacctgctgc aggcggagcc gtgcatacaa gaggactgga tttcgcctgc 600
gacatctaca tctgggcccc tctggctgga acatgtggcg tgctgctgct gagcctggtc 660
atcaccctgt actgcagcct gaagcggggc agaaagaagc tgctgtacat ctttaagcag 720
cccttcatgc ggcccgtgca gaccacacaa gaggaagatg gctgctcctg cagattcccc 780
gaggaagaag aaggcggctg cgagctgaga gtgaagttca gccgttctgc cgacgctccc 840
gcctataagc agggacagaa ccagctgtac aacgagctga acctggggag aagagaagag 900
tacgacgtgc tggacaagcg gagaggcaga gatcctgaga tgggcggcaa gcccagacgg 960
aagaatcctc aagagggcct gtataatgag ctgcagaaag acaagatggc cgaggcctac 1020
agcgagatcg gaatgaaggg cgagcgcaga agaggcaagg gacacgatgg actgtaccag 1080
ggcctgagca ccgccaccaa ggatacctat gatgccctgc acatgcaggc cctgcctcca 1140
agatcaggct ctggttctgg cagcggcagc atggtgtcta aaggcgagga actgttcacc 1200
ggcgtggtgc ccattctggt ggaactggac ggggatgtga acggccacaa gtttagcgtt 1260
agcggcgaag gcgaagggga tgccacatac ggaaagctga ccctgaagtt catctgcacc 1320
accggcaagc tgcctgtgcc ttggcctaca ctggtcacca cactgacata cggcgtgcag 1380
tgctttagca gataccccga ccatatgaag cagcacgact tcttcaagtc cgccatgcct 1440
gagggctacg tgcaagagcg gaccatcttc tttaaggacg acggcaacta caagaccagg 1500
gccgaagtga agtttgaggg cgacaccctg gtcaaccgga tcgagctgaa gggcatcgac 1560
ttcaaagagg atggcaacat cctgggccac aagctcgagt acaactacaa cagccacaac 1620
gtgtacatca tggccgacaa gcagaagaac ggcatcaagg ccaacttcaa gatccggcac 1680
aacatcgagg acggcagcgt tcagctggcc gatcactacc agcagaacac ccctatcgga 1740
gatggccctg tgctgctccc cgacaatcac tacctgagca cacagagcgc cctgagcaag 1800
gaccccaacg agaagaggga tcacatggtg ctgctggaat ttgtgaccgc cgcaggcatc 1860
accctcggca tggacgaact gtacaaa 1887
<210> 153
<211> 629
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 NKG2D.YA_CD8铰链TM_4-1BB_CD3ζ_EGFP 完全嵌合抗原受体
<400> 153
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro
145 150 155 160
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
165 170 175
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
180 185 190
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
195 200 205
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
210 215 220
Cys Ser Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
225 230 235 240
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
245 250 255
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
260 265 270
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
275 280 285
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
290 295 300
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
305 310 315 320
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
325 330 335
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
340 345 350
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
355 360 365
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
370 375 380
Gly Ser Gly Ser Gly Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr
385 390 395 400
Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His
405 410 415
Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys
420 425 430
Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp
435 440 445
Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg
450 455 460
Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro
465 470 475 480
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn
485 490 495
Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn
500 505 510
Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu
515 520 525
Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met
530 535 540
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
545 550 555 560
Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
565 570 575
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
580 585 590
Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
595 600 605
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
610 615 620
Asp Glu Leu Tyr Lys
625
<210> 154
<211> 1887
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码NKG2D.YA_CD8铰链TM_4-1BB_CD3ζ_EGFP 完全嵌合抗原受体
<400> 154
atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60
cctctgttca atcaagaggt gcagatccct ctgaccgaga gctactgtgg cccctgtcct 120
aagaactgga tctgctacaa gaacaactgc taccagttct tcgacgagag caagaattgg 180
tacgagagcc aggccagctg catgagccag aatgccagcc tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtcaag agcgcccact ggatgggact cgtgcacatc 300
cctacaaacg gcagctggca gtgggaggac ggctctatcc tgtctcctaa cctgctgacc 360
atcatcgaga tgcagaaggg cgactgcgcc ctgtacgcca gcagctttaa gggctacatc 420
gagaactgca gcacccctaa cacctacatc tgtatgcagc ggaccgtgac caccacacca 480
gctcctagac ctccaactcc tgctcctaca atcgccagcc agcctctgtc tctgaggcca 540
gaagcttgta gacctgctgc aggcggagcc gtgcatacaa gaggactgga tttcgcctgc 600
gacatctaca tctgggcccc tctggctgga acatgtggcg tgctgctgct gagcctggtc 660
atcaccctgt actgcagcct gaagcggggc agaaagaagc tgctgtacat ctttaagcag 720
cccttcatgc ggcccgtgca gaccacacaa gaggaagatg gctgctcctg cagattcccc 780
gaggaagaag aaggcggctg cgagctgaga gtgaagttca gccgttctgc cgacgctccc 840
gcctataagc agggacagaa ccagctgtac aacgagctga acctggggag aagagaagag 900
tacgacgtgc tggacaagcg gagaggcaga gatcctgaga tgggcggcaa gcccagacgg 960
aagaatcctc aagagggcct gtataatgag ctgcagaaag acaagatggc cgaggcctac 1020
agcgagatcg gaatgaaggg cgagcgcaga agaggcaagg gacacgatgg actgtaccag 1080
ggcctgagca ccgccaccaa ggatacctat gatgccctgc acatgcaggc cctgcctcca 1140
agatcaggct ctggttctgg cagcggcagc atggtgtcta aaggcgagga actgttcacc 1200
ggcgtggtgc ccattctggt ggaactggac ggggatgtga acggccacaa gtttagcgtt 1260
agcggcgaag gcgaagggga tgccacatac ggaaagctga ccctgaagtt catctgcacc 1320
accggcaagc tgcctgtgcc ttggcctaca ctggtcacca cactgacata cggcgtgcag 1380
tgctttagca gataccccga ccatatgaag cagcacgact tcttcaagtc cgccatgcct 1440
gagggctacg tgcaagagcg gaccatcttc tttaaggacg acggcaacta caagaccagg 1500
gccgaagtga agtttgaggg cgacaccctg gtcaaccgga tcgagctgaa gggcatcgac 1560
ttcaaagagg atggcaacat cctgggccac aagctcgagt acaactacaa cagccacaac 1620
gtgtacatca tggccgacaa gcagaagaac ggcatcaagg ccaacttcaa gatccggcac 1680
aacatcgagg acggcagcgt tcagctggcc gatcactacc agcagaacac ccctatcgga 1740
gatggccctg tgctgctccc cgacaatcac tacctgagca cacagagcgc cctgagcaag 1800
gaccccaacg agaagaggga tcacatggtg ctgctggaat ttgtgaccgc cgcaggcatc 1860
accctcggca tggacgaact gtacaaa 1887
<210> 155
<211> 629
<212> PRT
<213> 人工序列
<220>
<223> 合成肽 NKG2D.AF_CD8铰链TM_4-1BB_CD3ζ_EGFP 完全嵌合抗原受体
<400> 155
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro
145 150 155 160
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
165 170 175
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
180 185 190
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
195 200 205
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
210 215 220
Cys Ser Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
225 230 235 240
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
245 250 255
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
260 265 270
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
275 280 285
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
290 295 300
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
305 310 315 320
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
325 330 335
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
340 345 350
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
355 360 365
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
370 375 380
Gly Ser Gly Ser Gly Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr
385 390 395 400
Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His
405 410 415
Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys
420 425 430
Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp
435 440 445
Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg
450 455 460
Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro
465 470 475 480
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn
485 490 495
Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn
500 505 510
Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu
515 520 525
Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met
530 535 540
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
545 550 555 560
Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
565 570 575
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
580 585 590
Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
595 600 605
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
610 615 620
Asp Glu Leu Tyr Lys
625
<210> 156
<211> 1887
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码NKG2D.AF_CD8铰链TM_4-1BB_CD3ζ_EGFP 完全嵌合抗原受体
<400> 156
atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60
cctctgttca atcaagaggt gcagatccct ctgaccgaga gctactgtgg cccctgtcct 120
aagaactgga tctgctacaa gaacaactgc taccagttct tcgacgagag caagaattgg 180
tacgagagcc aggccagctg catgagccag aatgccagcc tgctgaaggt gtacagcaaa 240
gaggaccagg atctgctgaa gctggtcaag agcgcccact ggatgggact cgtgcacatc 300
cctacaaacg gcagctggca gtgggaggac ggctctatcc tgtctcctaa cctgctgacc 360
atcatcgaga tgcagaaggg cgactgcgcc ctgtacgcca gcagctttaa gggcttcatc 420
gagaactgca gcacccctaa cacctacatc tgtatgcagc ggaccgtgac caccacacca 480
gctcctagac ctccaactcc tgctcctaca atcgccagcc agcctctgtc tctgaggcca 540
gaagcttgta gacctgctgc aggcggagcc gtgcatacaa gaggactgga tttcgcctgc 600
gacatctaca tctgggcccc tctggctgga acatgtggcg tgctgctgct gagcctggtc 660
atcaccctgt actgcagcct gaagcggggc agaaagaagc tgctgtacat ctttaagcag 720
cccttcatgc ggcccgtgca gaccacacaa gaggaagatg gctgctcctg cagattcccc 780
gaggaagaag aaggcggctg cgagctgaga gtgaagttca gccgttctgc cgacgctccc 840
gcctataagc agggacagaa ccagctgtac aacgagctga acctggggag aagagaagag 900
tacgacgtgc tggacaagcg gagaggcaga gatcctgaga tgggcggcaa gcccagacgg 960
aagaatcctc aagagggcct gtataatgag ctgcagaaag acaagatggc cgaggcctac 1020
agcgagatcg gaatgaaggg cgagcgcaga agaggcaagg gacacgatgg actgtaccag 1080
ggcctgagca ccgccaccaa ggatacctat gatgccctgc acatgcaggc cctgcctcca 1140
agatcaggct ctggttctgg cagcggcagc atggtgtcta aaggcgagga actgttcacc 1200
ggcgtggtgc ccattctggt ggaactggac ggggatgtga acggccacaa gtttagcgtt 1260
agcggcgaag gcgaagggga tgccacatac ggaaagctga ccctgaagtt catctgcacc 1320
accggcaagc tgcctgtgcc ttggcctaca ctggtcacca cactgacata cggcgtgcag 1380
tgctttagca gataccccga ccatatgaag cagcacgact tcttcaagtc cgccatgcct 1440
gagggctacg tgcaagagcg gaccatcttc tttaaggacg acggcaacta caagaccagg 1500
gccgaagtga agtttgaggg cgacaccctg gtcaaccgga tcgagctgaa gggcatcgac 1560
ttcaaagagg atggcaacat cctgggccac aagctcgagt acaactacaa cagccacaac 1620
gtgtacatca tggccgacaa gcagaagaac ggcatcaagg ccaacttcaa gatccggcac 1680
aacatcgagg acggcagcgt tcagctggcc gatcactacc agcagaacac ccctatcgga 1740
gatggccctg tgctgctccc cgacaatcac tacctgagca cacagagcgc cctgagcaag 1800
gaccccaacg agaagaggga tcacatggtg ctgctggaat ttgtgaccgc cgcaggcatc 1860
accctcggca tggacgaact gtacaaa 1887

Claims (16)

1.一种天然NKG2D配体分子的非天然的、修饰的α1-α2结构域，其包括与所述天然α1-α2结构域至少80％的同一性，其中所述修饰的α1-α2结构域有超过5个所述天然α1-α2结构域的氨基酸被替换，并且其中所述修饰增加其与非天然NKG2D受体胞外域的结合亲和力，其中所述非天然NKG2D受体的所述胞外域对至少一种天然NKG2D配体的亲和力小于所述天然NKG2D受体胞外域对所述至少天然NKG2D配体的所述亲和力。
2.根据权利要求1所述的非天然的、修饰的α1-α2结构域，其中，所述至少一种天然NKG2D配体是SEQ ID NO:1-9中的任一种。
3.根据权利要求1所述的非天然的、修饰的α1-α2结构域，其中，从所述天然α1-α2结构域替换的所述氨基酸在SEQ ID NO:4的8、80、154、155、156、157、158和159位中的三个或更多个位置处。
4.根据权利要求3所述的非天然的，修饰的α1-α2结构域，其包括选自于由SEQ ID NO:111、113、115和117组成的组的氨基酸序列。
5.根据权利要求1所述的非天然的、修饰的α1-α2结构域，其中，所述非天然NKG2D受体胞外域包括SEQ ID NO:17的氨基酸序列，但其中SEQ ID NO:17的第75位和第122位处的所述酪氨酸均已被另一氨基酸替代。
6.根据权利要求5所述的非天然的、修饰的α1-α2结构域，其中，替代第75位的所述酪氨酸的所述氨基酸是丙氨酸，并且其中替代第122位处的所述酪氨酸的所述氨基酸是苯丙氨酸。
7.根据权利要求1所述的非天然的、修饰的α1-α2结构域，还包括连接的原子或连接的异源分子，从而产生双特异性分子。
8.根据权利要求7所述的非天然的、修饰的α1-α2结构域，其中，所述异源分子是肽或多肽。
9.根据权利要求8所述的非天然的、修饰的α1-α2结构域，其中，所述多肽是抗体、抗体片段、细胞因子、淋巴因子或激素。
10.根据权利要求7所述的非天然的、修饰的α1-α2结构域，其中，所述异源分子是寡糖、树枝状大分子、扭结菌素、激素、核酸或脂质。
11.一种哺乳动物细胞，其附着有根据权利要求1所述的非天然NKG2D受体胞外域。
12.根据权利要求11所述的哺乳动物细胞，其中，所述哺乳动物细胞是淋巴细胞或巨噬细胞。
13.根据权利要求12所述的哺乳动物细胞，其中，所述哺乳动物细胞是人淋巴细胞或人巨噬细胞。
14.根据权利要求7所述的非天然的、修饰的α1-α2结构域，其中，当所述非天然的、修饰的α1-α2结构域与具有连接于其上的非天然NKG2D受体胞外域的哺乳动物细胞接触时，所述原子或异源分子被递送至所述哺乳动物细胞。
15.根据权利要求14所述的非天然的、修饰的α1-α2结构域，其表现出对表达所述非天然NKG2D胞外域的细胞的活化增强，导致所述细胞表现出比将天然NKG2D配体或具有天然或原生α1-α2结构域的双特异性分子递送至所述细胞更高的靶细胞杀伤效力。
16.根据权利要求14所述的非天然的、修饰的α1-α2结构域，其表现出比表达天然NKG2D胞外域的细胞更高的对表达所述非天然NKG2D胞外域的细胞的活化，导致表达所述非天然NKG2D胞外域的上述细胞表现出比表达上述天然NKG2D胞外域的细胞更高的靶细胞杀伤效力。
CN201980022711.XA 2018-03-27 2019-03-27 结合非天然NKG2D受体的非天然NKG2D配体的修饰的α1-α2结构域 Pending CN112074532A (zh)

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