CN113614103A - 不直接向其附着的细胞传导信号的非天然nkg2d受体 - Google Patents

不直接向其附着的细胞传导信号的非天然nkg2d受体 Download PDF

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CN113614103A
CN113614103A CN201980087716.0A CN201980087716A CN113614103A CN 113614103 A CN113614103 A CN 113614103A CN 201980087716 A CN201980087716 A CN 201980087716A CN 113614103 A CN113614103 A CN 113614103A
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卡曼·金姆
大卫·W·小马丁
史蒂文·威廉姆斯
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Xyphos Biosciences Inc
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Abstract

本公开涉及附着于哺乳动物细胞表面的非天然NKG2D受体，其中当所述非天然受体被经修饰以特异性结合非天然NKG2D受体的NKG2D配体的同源非天然α1‑α2结构域结合时，所述非天然受体不直接传导信号给所述细胞或直接激活所述细胞。NKG2D配体的非天然α1‑α2结构域可附着于包括多肽的异源原子或异源分子，在一些实施方式中，所述异源原子或异源分子为细胞因子或经修饰的细胞因子、抗体或抗体的片段。

Description

不直接向其附着的细胞传导信号的非天然NKG2D受体
技术领域
本申请总体上涉及附着于哺乳动物细胞的非天然NKG2D受体的非天然胞外域，其中当与经修饰以特异性结合非天然NKG2D受体的非天然NKG2D配体结合时，所述受体不直接激活或直接传导信号给所述哺乳动物细胞，并且涉及附着到NKG2D配体的经修饰的α1-α2结构域的异源分子。
背景技术
NKG2D是在自然杀伤(NK)细胞以及某些T细胞和巨噬细胞表面表达为II型同型二聚体整合蛋白的活化性受体。当与主要在受损细胞表面表达的其八种天然配体之一结合时，NKG2D激活NK细胞以杀伤受应激的细胞，或者当在T细胞上时，配体占据的NKG2D共刺激激活的T细胞以实现其效应子功能。已经对人类天然NKG2D的胞外域、数种其可溶性天然配体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求解了三维结构。NKG2D配体的单体α1-α2结构域特异性结合至天然NKG2D同型二聚体的两个胞外域。
发明内容
本公开涉及附着于哺乳动物细胞表面的非天然NKG2D受体，其中当所述非天然受体与经修饰以特异性结合该非天然NKG2D受体的NKG2D配体的同源非天然α1-α2结构域结合时，所述非天然受体不直接传导信号给或直接激活所述细胞。NKG2D配体的非天然α1-α2结构域可附着至异源原子或包括多肽在内的异源分子，在一些实施方式中，所述多肽为细胞因子或经修饰的细胞因子、抗体或抗体片段。细胞的直接活化或向细胞的直接信号传导并非由附着的非天然NKG2D受体所介导，并且即使当免疫突触已经出现时也不发生细胞的直接活化或向细胞的直接信号传导。
附图说明
图1A-1B：(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。指示了Y152和Y199的位置，并且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(SEQ ID NO:4)和ULBP2(包括ULBP2.R80W(SEQ ID NO:108))的非天然变体的α1-α2结构域的比对。灰色突出显示的是对非天然ULBP2变体与非天然NKG2D.YA或NKG2D.AF受体结合至关重要的残基。指示了残基R80以及M154-F159区域的位置，其用于研究与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 IDNO.:115；和ULBP2.AB，SEQ ID NO.:117)结合的正交变体。
图2：在Akta HiLoad 16/600Superdex 200柱上分析的非天然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证，而聚集的材料在较低体积下更快地洗脱。通过氨基酸编号Y152、Y199或两者来指示修饰的位点和性质(从图的顶部开始，SEQ ID NO:48，43，42，58，41和40)。
图3：在Akta Superdex 200Increase 10/300GL柱上分析了具有一个或两个氨基酸改变的非天然Fc-eNKG2D变体的尺寸排阻色谱特性。正确组装的材料的迁移通过在较高体积洗脱的离散对称峰来例证，而聚集的材料(特征在于低振幅宽峰或一系列峰)在较低体积下洗脱。括号中的字母分别表示位置152和199处的氨基酸(从顶部开始按顺序为SEQ IDNO:57，56，55，54，53，52，51，50，49，48，47，46，45，44和40)。
图4：ULBP2野生型、MICwed-和MIC25-利妥昔单抗MicAbody与Fc-eNKG2D候选物的ELISA结合。关键字在图的顶部示出，但是由于许多曲线重叠，所以在每个图中也标记了单独的曲线。
图5：eNKG2D变体与野生型配体的结合。野生型配体(都是Fc-融合形式)被OctetAHC生物传感器捕获，并且每种天然NKG2D、NKG2D.Y152A或eNKG2D5(Y152A/Y199F)作为Fc融合体从300nM滴定至0.41nM。通过Octet量化最大结合响应。(注意每个图表的不同纵坐标)。
图6：各个噬菌体变体的滴定ELISA以确认与Fc-NKG2D.AF的选择性结合和与Fc-NKG2D.wt结合的减少或消除。在图22中详细描述了突变。
图7：结合NKG2D.wt、NKG2D.YA和NKG2D.AF的四种非天然α1-α2ULBP2变体MicAbody的ELISA数据。将Fc-NKG2D变体用作捕获剂。用HRP-缀合的抗人κ滴定并检测MicAbody。
图8：用NetMHC4.0服务器，针对HLA超型代表进行查询，检查了ULBP2.C(SEQ IDNO:111)、ULBP2.R(SEQ ID NO:113)、ULBP2.AA(SEQ ID NO:115)和ULBP2.AB(SEQ ID NO:117)相对于野生型ULBP2(SEQ ID NO:4)的肽-MHCI免疫原性的变化。对于输入序列，输入每个变体的可变区(根据图1B中的比对，残基154-159)以及上游和下游的九个残基(总共24个残基)，并检查9-聚体肽窗以预测免疫原性。暗灰色方框对应于强烈预测结合MHCI口袋(定义为具有％秩<0.5)并且因此具有强烈被呈递机会的肽。浅灰色方框对应于预测的弱结合剂(％秩＜2)。附加细节参见实施例5正文。
图9：使用NetMHCII 2.3服务器，针对HLA-DR、HLA-DQ、HLA-DP进行查询，检查了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)相对于野生型ULBP2(SEQ ID NO:4)的肽-MHC II类免疫原性的变化。对于输入序列，输入每个变体的可变区(根据图1B中的比对，残基154-159)以及上游和下游的15个残基(总共36个残基)，并且检查了15-聚体的肽窗以预测免疫原性。暗灰色方框对应于强烈预测结合MHCII口袋并因此可能被呈递且具有免疫原性的肽。浅灰色方框对应于预测的弱结合剂。
图10A-10B：ELISA-测量的包含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)的利妥昔单抗-MicAbody与天然NKG2D.wt、NKG2D.YA和NKG2D.AF的结合。(图10A)ELISA曲线。对于一些在较高浓度下的测定，458nm吸收的降低是假象，在较高浓度的高亲和力接合体中经常见到这种假象，这是由于TMB-ultra ELISA显影试剂的沉淀引起的。(图10B)EC50值(以nM报告)，如在GraphPad Prism中基于(A)中的曲线测定的。nd＝未确定，因为增加的浓度和结合之间缺乏关系。
图11A-11C：用未转导或用NKG2D.wt、NKG2D.YA或NKG2D.AF CAR构建体转导的CD8效应细胞进行体外细胞溶解测定，所述NKG2D.AF CAR构建体由CD8a铰链/跨膜结构域和细胞内4-1BB和CD3ζ信号传导结构域组成。靶细胞预先负载钙黄绿素，并以递增的效应物与靶比率(E:T)暴露于效应细胞。五小时后定量释放的钙黄绿素。(图11A)HeLa细胞的细胞溶解，(图11B)转染过表达表面ULBP1的HeLa细胞的溶解，和(图11C)在表面上表达非天然ULBP2.R的HeLa细胞的细胞溶解。误差线对应于实验中的技术重复的标准偏差。
图12A-12B：MicAbody引起NKG2D-CAR CD8T细胞对肿瘤系的细胞溶解。(图12A)将Ramos细胞(其表达CD20，利妥昔单抗的靶标)预先加载钙黄绿素，并且以20:1的E:T比暴露于NKG2D.AF-或NKG2D.YA-CAR细胞，同时增加ULBP2.S3或ULBP2.R利妥昔单抗-Micabody的浓度。共孵育两小时后定量细胞溶解水平。(图12B)将转染以表达人类Her2的小鼠肿瘤系CT26用作与Ramos细胞平行的细胞溶解靶。用饱和浓度(5nM)的利妥昔单抗-ULBP2.R、曲妥珠单抗-ULBP2.R或两种MicAbody的等摩尔混合物预装备NKG2D.AF-CAR CD8 T细胞。通过洗涤除去未结合的MicAbody，并将CD8细胞以两个不同的E:T比率添加到靶细胞。两小时后测量细胞溶解。
图13A-13D：潜在MicAbody和MicAdaptor形式的说明。(图13A)在开发MicAbody和MicAdaptor试剂中利用的各种抗体Fc变体，并且包括(a)野生型人类IgG1 Fc，(b)使得FcADCC-缺陷的两个突变，和(c)在每个Fc-Fc1或Fc2-(其允许产生异二聚体)-Fc分子中的静电转向突变，该分子还含有ADCC-缺陷突变。(图13B)正交配体如何可以与(a)重链或(b)轻链的C-末端融合以产生MicAbody试剂的实例。(图13C)具有直接正交配体融合而没有抗体组分的MicAdaptor实例。(图13D)说明了多种MicAdaptor分子，其可以在人类IgG1 Fc的背景下产生以增强血清稳定性，并且可以(a，b，c；在文本和图例中表示为“Fc1/Fc2”)或可以不包括(d，e)异二聚体-Fc突变，这取决于异源载物或正交配体的所需分子的化合价。这也可以包括根据所需的载物、化合价和功能(f,g)利用完整的抗体结构，并且可以是重链或轻链融合体。
图14：CAR构建体、沉默CAR和其它CAR变体的示意图。如所示的每个构建体的SEQID NO。
图15A-15C：选择性递送细胞因子融合体至NKG2D.YA-CAR表达CD8 T细胞。(图15A)将表达NKG2D.wt-CAR(SEQ ID NO:151)或NKG2D.YA-CAR SEQ ID NO:153)的CD8细胞暴露于30或300IUe/mL重组人类IL2(rhIL2)或IL15(rhIL15)，或变异的突变体-IL2/-IL15正交配体直接融合体或异源二聚体Fc(Fc1/Fc2)背景下的融合体。三天后，通过WST细胞增殖试剂定量增殖。此处的数据显示为相对于无细胞因子对照归一化。(图15B)将用编码NKG2D.YA-CAR的载体转导的人类T细胞暴露于各种配体-细胞因子融合分子七天，并随时间跟踪培养物中GFP+CAR-细胞的百分比。(图15C)如WST测定所确定的，正交配体增强了IL21和突变体-IL21试剂向NKG2D.YA-CAR细胞的递送，并且在三天的培养中促进了它们相对于未转导的细胞的扩增。横坐标图例的括号中指示了细胞因子或细胞因子-MicAdaptor的IUe/mL。MicAdaptor SEQ ID NO参见图24。
图16A-16C：探索单独的NKG2D.YA胞外域促进正交配体-细胞因子-融合体递送至细胞的充分性数据。(图16A)单独的NKG2D.YA胞外域(SEQ ID NO:157)不能引起利妥昔单抗-ULBP2.S3 MicAbody(SEQ ID NO:98和129)杀伤Ramos细胞。(图16B)暴露于各种细胞因子试剂三天后的WST增殖测定。(图16C)WST增殖测定证明了NKG2D.YA-CAR与正交配体的接合，但不存在融合的细胞因子或细胞因子突变体时不足以驱动细胞扩增。MicAdaptor SEQID NO参见图24。横坐标图例的括号中指示了细胞因子和细胞因子-MicAdaptor的IUE/mL量。
图17A-17C：(图17A)在用指定的细胞因子或细胞因子-MicAdaptor一起孵育三天后，各种共刺激结构域突变体(SEQ ID NO:161，163和165)的WST增殖测定。(图17B)检验了相同的共刺激结构域CAR突变体在利妥昔单抗-ULBP2.S3 MicAbody(SEQ ID NO:98和129)存在下有效裂解负载钙黄绿素的Ramos靶细胞的能力。(图17C)如WST测定法所评估的，在与细胞因子试剂孵育三天后，NKG2D.YA胞外域(NKG2D.YA-ecd)与完整CD19scFv-CAR的共表达足以促进增殖。MicAdaptor SEQ ID NO参见图24。
图18：通过尺寸排阻色谱(SEC)确定候选的非天然Fc-eNKG2D变体突变和蛋白质聚集性质的汇总。
图19：通过MICwed-MicAbody或MIC25-MicAbody归一化至野生型NKG2D结合的eNKG2D变体的饱和百分比(Rmax)。野生型Fc-NKG2D和每种Fc-eNKG2D受体被AHC生物传感器捕获，然后暴露于20nM的曲妥珠单抗-特异性MicAbody。监测解离动力学，并对Fc-eNKG2D融合体的Rmax值进行排名。那些未测试的样品(nt)是由于SEC分级后表达或回收的材料的严重聚集或不足量导致的。
图20：图3显示的Fc-eNKG2D ELISA的EC50值(nM)。nt＝未测试；nb＝即使在300nM下也没有结合或非常低的结合，因此未计算EC50值。
图21：通过spot ELISA证实了ULBP2内组合突变的亚集，其导致噬菌体克隆与NKG2D.AF和天然NKG2D.wt有选择性结合。通过在所选噬菌体中出现的频率对突变体进行排名。
图22：通过定量ELISA，利妥昔单抗-MicAbody形式的NKG2D.AF选择的ULBP2变体保留了它们与NKG2D.AF的结合特异性。显示了每种ULBP2变体的特定氨基酸修饰，均以其与Fc-NKG2D.wt融合的结合相对于Fc-NKG2D.AF融合的比率表示。ULBP2的氨基酸残基位置是图1B的那些。
图23：在ULBP2.R80W(图1B；SEQ ID NO:108)的指定氨基酸位置处的选择突变，其产生Y152A-特异性噬菌体克隆。
图24：MicAdaptor SEQ ID NO和从瞬时转染中纯化的方法。
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细胞在人类和其它哺乳动物中作为抵抗肿瘤和感染细胞的首要的先天性防御而具有重要作用(Cerwenka,A.,andL.L.Lanier.2001.NK cells,viruses and cancer.Nat.Rev.Immunol.1:41-49)。NK细胞和某些T细胞在其表面上表现出NKG2D，这是一种突出的同源二聚体表面免疫受体，其负责识别靶细胞并激活抵抗病理细胞的先天性防御(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)。人类NKG2D分子具有结合其八种不同的同源配体的C型凝集素样细胞外(胞外-)结构域，研究最多的配体是84％序列相同或同源的单体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(Weiset 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的非病理性表达限于某些肠上皮、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响应于多种类型的细胞应激(如增殖、氧化和热休克)而发生，并将细胞标记为病理性的(Groh et al.1996.Cell stress-regulated human MHCclass I gene 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涉及一些自身免疫疾病(Ravetch,JVand Lanier LL.2000.Immune Inhibitory Receptors.Science 290:84-89；Burgess,SJ.2008.Immunol.Res.40:18-34)。NKG2D配体(如多态性MICA和MICB)的差异调节，对于为免疫系统提供一种识别和响应各种紧急提示的手段并同时仍然保护健康细胞免受有害攻击是重要的(Stephens HA,(2001)MICA and MICB genes:can the enigma of theirpolymorphism be resolved？Trends Immunol.22:378-85；Spies,T.2008.Regulation ofNKG2D ligands:a purposeful but delicate affair.Nature Immunol.9:1013-1015)。
病毒感染是MIC蛋白表达的常见诱导剂，可识别病毒感染的细胞以用于NK或T细胞攻击(Groh et al.1998；Groh et al.2001.Co-stimulation of CD8+αβTcells by NKG2Dvia engagement 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，巨细胞病毒和其它病毒已经进化了防止MIC蛋白在它们感染的细胞表面上表达的机制，以逃避被先天免疫系统靶向(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 BDA0003145238650000081
Cekinovic,E Markova Car,EP Pugel,A Cikovic,VJLisnic,WJ Britt,UKoszinowski,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)。
尽管它们的应激，许多恶性细胞，例如肺癌和成胶质细胞瘤脑癌的那些细胞，也避免MIC蛋白的表达，并且结果可能是特别具有侵略性的，因为它们也逃避了先天免疫系统(Busche,A et al.2006,NK cell mediated rejection of experimental human lungcancer by genetic over expression of MHC class I chain-related gene A.HumanGene Therapy 17:135-146；Doubrovina,ES,MM Doubrovin,E Vider,RB Sisson,RJ O’Reilly,B Dupont,and YM Vyas,2003.Evasion from NK Cell Immunity by MHC Class IChain-Related Molecules Expressing Colon Adenocarcinoma(2003)J.Immunology6891-99；Friese,M.et al.2003.MICA/NKG2D-mediated immunogene therapy ofexperimental gliomas.Cancer Research 63:8996-9006；Fuertes,MB,MV Girart,LLMolinero,CI Domaica,LE Rossi,MM Barrio,J Mordoh,GA Rabinovich and NW Zwirner.(2008)Intracellular Retention of the NKG2D Ligand MHC Class I Chain-RelatedGene A in Human Melanomas Confers Immune Privilege and Prevents NK Cell-Mediated Cytotoxicity.J.Immunology,180:4606-4614)。
与NKG2D结合的人类MICA的高分辨率结构已被解析，并证明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；蛋白质数据库接入代码1HYR)。MICA的α3结构域，如MICB的α3结构域，通过短的柔性的连接肽与α1-α2平台结构域连接，并且其本身作为“间隔区”天然位于所述平台和MIC表达细胞的表面之间。人类MICA和MICBα3结构域的三维结构几乎相同(在94C-αα上，
Figure BDA0003145238650000091
Figure BDA0003145238650000092
)且在功能上可互换(Holmes et al.2001.Structural Studies ofAllelic Diversity of the MHC Class I Homolog MICB,a Stress-Inducible Ligandfor the Activating Immunoreceptor NKG2D.J Immunol.169:1395-1400)。
T细胞、NK细胞和巨噬细胞可以使用基因转移技术进行修饰，以在它们的表面上直接和稳定地表达赋予新型抗原特异性的抗体的结合结构域(Saar Gill&CarlH.June.Going viral:Chimeric Antigen Receptor(CAR)T cell therapy forhematological malignancies.Immunological Reviews 2015.Vol.263:68–89；WolfgangGlienke,Ruth Esser,Christoph Priesner,Julia D.Suerth,Axel Schambach,WinfriedS.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)。CAR-T细胞是这种将特异性抗体的抗原识别结构域与CD3-ζ链的融合胞内结构域组合的方法的应用。CD3-ζ链是信号从内源性T细胞受体(TCR)的胞外域向胞内空间的主要传递者。用CD3-ζ链和共刺激分子(如CD27、CD28、ICOS、4-1BB或OX40)构建的CAR在结合靶抗原后触发CAR-T细胞活化，其方式类似于内源性T细胞受体但不依赖于主要组织相容性复合物(MHC)。
已经描述了NKG2D配体的某些非天然α1-α2结构域，其经修饰以比天然α1-α2结构域更高的亲和力结合天然人类NKG2D受体(Candice S.E.Lengyel,Lindsey J.Willis,Patrick Mann,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 2012VOL 21:1396—1402)。在本文中，我们描述了如下形式的非天然NKG2D受体与哺乳动物细胞表面的附着，其保留经修饰的非天然NKG2D配体与附着的异源分子的特异性结合，但即使在该哺乳动物细胞与异源分子所靶向的细胞或其它表面形成免疫突触时，所述非天然受体也可避免直接或顺式激活哺乳动物细胞或向哺乳动物细胞进行细胞内信号传导。非天然NKG2D受体本身已在一个或两个特定位点发生了突变，其中每一种突变均导致与NKG2D配体的所有天然α1-α2结构域的结合受损或丧失(David J.Culpepper,Michael K.Maddox,Andrew B.Caldwell,and Benjamin J.McFarland.Systematic mutation and thermodynamic analysis ofcentral tyrosine pairs in polyspecific NKG2D receptor interactions.MolImmunol.2011January；48(4):516–523；USPTO申请14/562,534；USPTO临时申请62/088,456))。本发明产生了CAR，当其附着于哺乳动物细胞表面时，提供了沉默受体，其可以作为替代高亲和力受体，用于异源原子或异源分子向细胞表面的附着。因此，通过对非天然修饰的NKG2D受体具有特异性的附着的非天然配体，异源分子(例如包含缺陷性细胞因子)可以被特异性地递送到哺乳动物细胞表面的沉默受体，而不被递送到缺乏同源沉默受体的细胞。一旦结合到带有沉默受体的细胞上，缺陷性异源分子可结合到细胞表面上保留了与其结合性的其各自受体亚基上，从而直接向细胞传导信号，如同细胞被野生型配体刺激一样。
当然，在哺乳动物细胞上包含惰性非天然NKG2D、CD3-ζ和共刺激结构域(如CD28、4-1BB、ICOS或OX40)的CAR能够在形成免疫突触后直接刺激并激活CAR-细胞。这种第二或第三代CAR-T细胞的活化依赖于其CD3-ζ结构域的功能和至少一个共刺激结构域(例如4-1BB或CD28)的功能。然而，作为沉默CAR，这种CAR可以作为替代高亲和力受体，用于结合同源非天然配体-附着的异源分子，该异源分子与其各自天然受体或受体亚基具有缺陷性结合。这种高亲和力结合使得附着于非天然配体的异源分子能够通过其各自的其它受体亚基将信号传递至细胞，所述其它受体亚基保留了结合性。
重要的是，当选择性失活这种具有直接活化能力的CAR的CD3-ζ结构域时，其仍可作为沉默CAR，并使与其各自的天然受体或受体亚基具有缺陷性结合的同源非天然配体-附着的异源分子能够经由它们各自的其它受体亚基向细胞传递信号。当CAR共刺激结构域(如4-1BB)被失活并且保留活性CD3-ζ结构域时，CAR不能作为沉默受体。即，尽管CD3-ζ不是必需的，但需要功能性共刺激结构域以使异源分子(如附着于结合至受体的同源非天然配体的缺陷性细胞因子)能够介导其各自向CAR细胞的信号传导。
本发明揭示了为使附着至同源非天然配体的异源缺陷性细胞因子能够介导其各自向CAR细胞的信号传导，出乎意料的需要共刺激结构域而非CD3-ζ。此外，本发明公开了共刺激结构域可以顺式或反式作用于沉默受体，该沉默受体上附着有融合至缺陷性异源缺陷分子的同源配体。
当靶向特定分子的异源分子(如抗体或抗体片段)附着至同源非天然NKG2D配体时(同源非天然NKG2D配体又附着至沉默受体)，携带沉默受体的哺乳动物细胞将归巢至靶向异源分子将其导向的表面。即使在携带沉默受体的细胞和靶细胞表面之间产生“突触”时，前者也不会被沉默受体激活。
因为在细胞表面上存在本发明的基于非天然NKG2D的沉默受体的许多拷贝，所以在生产过程或治疗方案中，通过异源分子的归巢和/或选择性激活可以是多重的或顺序改变的。
携带沉默受体CAR的细胞也可表达与沉默CAR正交的另一种受体或CAR，所述另一种受体或CAR独立于所述沉默CAR发挥作用，以在适当刺激时特异性地和直接地激活相同细胞或以其它方式向相同细胞传导信号。其它或“第二”正交CAR可以是传统的单链Fv(scFv)-CAR或具有其自身同源非天然α1-α2配体的第二正交的、基于非天然修饰的NKG2D的CAR。(AF临时参考)。产生带有多于一种正交非天然CAR(沉默的或活性的)和(具有附着的异源分子或原子的)多种同源非天然配体的免疫系统的效应细胞的能力，极大地扩展了过继性细胞疗法(ACT)的效用、灵活性和控制。
在表征细胞上的沉默CAR及其对顺式或反式作用共刺激结构域(如4-1BB)的依赖性的方法中，观察到与未修饰的人类T细胞相比，表达具有共刺激结构域的沉默CAR的人类T细胞表现出对天然IL-2或与天然或突变体IL-2融合的同源非天然配体的应答显著增强，其中所述突变体IL-2对其受体α-亚基具有低亲和力。这一观察结果在表达CAR的细胞的离体或体内优先扩增中具有重要效用，所述CAR包含共刺激结构域且有或没有CD3-ζ结构域。
如本文所用，“可溶性MIC蛋白”、“可溶性MICA”和“可溶性MICB”指MIC蛋白，其含有MIC蛋白的α1-α2结构域、有或没有MIC蛋白的α3结构域、但没有跨膜或细胞内结构域。NKG2D配体‘ULBP1-6’天然不具有α3结构域(Cerwenka A,Lanier LL.2004.NKG2D ligands:unconventional MHC class I-like molecules exploited by viruses andcancer.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”指结合NKG2D受体的配体的蛋白质结构域。
在一些实施方式中，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结构域具有至少80％同一性或同源性(SEQ ID NO:1-9分别为MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6和OMCP)。在其它实施方式中，经修饰的α1-α2结构域与NKG2D配体的原生或天然α1-α2结构域具有85％同一性。在另外其它实施方式中，经修饰的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90％同一性，并且结合非天然NKG2D。
优选地，本发明的非天然MIC蛋白的经修饰的或非天然α1-α2结构域与8种人类NKG2D配体蛋白(SEQ ID NO:1-8)之一的原生或天然α1-α2结构域具有至少80％同一性或同源性，并且结合非天然NKG2D胞外域。在一些实施方式中，所述非天然α1-α2结构域与NKG2D配体蛋白的原生或天然α1-α2结构域具有85％同一性，并且结合非天然NKG2D。在其它实施方式中，所述非天然α1-α2平台结构域与人类天然α1-α2结构域蛋白的原生或天然α1-α2平台具有90％、95％、96％、97％、98％或99％同一性，并且结合非天然NKG2D。
在一些实施方式中，可将异源分子标签融合于可溶性MIC蛋白的非天然α1-α2结构域的N-末端或C-末端，或融合于附着的异源肽或蛋白的N-末端或C-末端，以协助可溶性配体的纯化。标签序列包括肽，例如聚组氨酸、myc-肽、FLAG标签、链霉亲和素样标签，或小分子，例如生物素。可在分离MIC分子后，通过本领域技术人员已知的方法可以除去这些标签。
可以在NKG2D配体的α1-α2结构域中进行特异性突变，以产生结合非天然NKG2D受体的非天然α1-α2结构域，所述受体本身被工程化以对天然NKG2D配体具有降低的亲和力。这可以通过例如遗传工程来完成。如此修饰的非天然NKG2D受体可用于在NK细胞、T细胞、巨噬细胞或免疫系统的其它细胞的表面上产生基于NKG2D的CAR，其可结合包含非天然α1-α2结构域的分子。如下所述，与目前的CAR-T细胞和CAR-NK细胞相比，这些非天然NKG2D受体及其同源非天然NKG2D配体可提供用于治疗癌症和病毒感染的重要安全性、功效和制造优势。当哺乳动物细胞表面上的非天然NKG2D受体的细胞内信号传导如在本发明中一样已被沉默时，这些发明的CAR可作为用于原本缺陷性异源分子(例如细胞因子、趋化因子、淋巴因子、细胞毒素和与正交NKG2D配体融合或缀合的原子)的替代高亲和力受体。这提供了异源分子向携带沉默受体的细胞的直接且特异性递送，而沉默受体本身并不直接激活其宿主细胞。此外，结合特定靶标并由此结合携带这些靶标的细胞或其它表面的异源分子，可为携带沉默受体的细胞提供特异性归巢功能，而不会使其非故意地激活或刺激。
包含不结合或仅很少结合天然NKG2D配体的非天然NKG2D受体的胞外域的CAR-T或CAR-NK细胞，不会受到任何天然配体的激活，因此不会像表达基于天然NKG2D受体的CAR的细胞那样具有产毒性。此外，细胞上非天然NKG2D受体的胞外域不会被可溶形式的或骨髓衍生抑制细胞(MDSC)上的天然NKG2D配体下调(Deng W,Gowen BG,Zhang L,Wang L,Lau S,Iannello A,Xu J,Rovis TL,Xiong N,Raulet DH,2015.Antitumor immunity.A shedNKG2Dligand that promotes natural killer cell activation and tumorrejection.Science.2015Apr 3；348(6230):136-9.doi:10.1126/science.1258867.Epub2015Mar 5)。然而，当这样的携带非天然NKG2D受体胞外域的CAR细胞被具有本发明同源非天然α1-α2结构域及其异源靶向基序的双特异性分子接合时，CAR将被激活并且表达CAR细胞的效应子功能，其中所述异源靶向基序已经被发现并结合其预期靶标。
因为除了在存在接合的双特异性分子时，所述双特异性分子包含同源非天然NKG2D受体胞外域，包含非天然NKG2D受体胞外域的CAR-T或CAR-NK细胞不被激活，因此它们的活化可以通过施用的双特异性分子控制，所述双特异性分子作为生物药物将表现出本领域公知的药代动力学和药效学。在不良事件发生的情况下，医师可以简单地改变施用的双特异性分子的给药方案，而不必像目前所做的那样必须使用诱导的自杀机制来破坏输注的CAR细胞(Monica Casucci and Attilio Bondanza.Suicide Gene Therapy to Increasethe Safety of Chimeric Antigen Receptor-Redirected T Lymphocytes.JCancer.2011；2:378–382)。此外，可以同时或顺序施用具有不同特异性靶向基序的此类双特异性分子，以协助解决由于靶抗原丧失所致的肿瘤抗性和逃脱，而不必创建、扩增和输注多种不同的自体CAR细胞(Gill&June，2015)。由于所有CAR构建体对于所有CAR细胞而言可以相同，并且靶向特异性简单地由施用的本发明的双特异性分子的靶向基序决定，因此制造过程可被简化并且更便宜。
作为附着于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、包含Ig折叠或Ig结构域的蛋白(包括募集天然分子或不能募集或结合天然分子的经修饰的Fc结构域)、球蛋白、白蛋白、纤连蛋白和纤连蛋白结构域、整联蛋白、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、用于细胞受体的病毒配体、激素、细胞因子和经修饰的细胞因子(如白细胞介素、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。也可以将非蛋白质分子(如多糖、树枝状聚合物、聚乙二醇、肽聚糖、抗生素和聚酮化合物)连接到NKG2D配体的经修饰的α1-α2结构域。
因此，本发明扩大了这种显著的、非常有前景的免疫方法的多样性和实用性，以用CAR-T细胞、CAR-NK细胞和CAR-巨噬细胞样细胞治疗癌症，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“蛋白质”可互换使用；“异源分子”、“异源肽”、“异源序列”或“异源原子”分别为不是在与主题分子物理结合中天然或正常发现的分子、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”、“原生”和“野生型”可互换使用，“NKG2D”和“NKG2D受体”可互换使用。本文的术语“抗体”以最广义使用，并且具体地涵盖单克隆抗体、多特异性抗体(例如双特异性抗体)和抗体片段，只要它们表现出期望的生物学活性即可。“抗体片段”包含抗体的一部分，优选包含其抗原结合区。抗体片段的实例包括Fab、Fab'、F(ab')2、Fv片段和可被插入的Fv；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。
术语“包含”与“包括”、“含有”或“特征在于”可互换使用，是包含性或开放式语言，并且不排除另外的未列举的要素或方法步骤。短语“由……组成”排除权利要求中未指定的任何要素、步骤或成分。短语“基本上由……组成”将权利要求的范围限制至指定的材料或步骤以及那些实质上不影响所要求保护的发明的基本和新颖特征的材料或步骤。本公开考虑了对应于这些短语中的每一个的范围的本发明组合物和方法的实施方式。因此，包含叙述要素或步骤的组合物或方法考虑了其中组合物或方法基本上由那些要素或步骤组成或由那些要素或步骤组成的特定实施方式。
本文引用的所有参考文献都通过引用整体并入本文，无论先前是否具体并入。如本文所用，术语“一(a)”、“一(an)”和“任何”均旨在包括单数和复数形式。
现在已经充分描述了本发明，本领域技术人员将理解，在不背离本发明的精神和范围并且在没有过度实验的情况下，可以在宽范围的等效参数、浓度和条件下实施本发明。尽管已经结合具体实施方式描述了本发明，但应当理解的是，能够进一步修改本发明。本申请旨在涵盖通常遵循本发明原理的对本发明的任何变化、用途或改编，并且包括在本发明所属领域内已知或习惯做法范围内并且可以应用于上文所述基本特征的与本公开内容的偏离。
实施例
经修饰的NKG2D受体胞外域和NKG2D配体的经修饰的α1-α2结构域
实施例1.将人类NKG2D受体的酪氨酸152修改为丙氨酸(Y152A)且将酪氨酸199修改为苯丙氨酸(Y199F)，以产生惰性NKG2D胞外域
已由他人证明，人类NKG2D中在酪氨酸152或在酪氨酸199的突变，相当于NKG2D胞外域的位置73和120(图1A,SEQ ID NO.:17)可以大大降低与天然配体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)。我们推论，鉴于任一酪氨酸残基的突变极大地影响了NKG2D结合其天然配体的能力，则在酪氨酸152(Y152)和酪氨酸199(Y199)上的同时突变将实际上消除受体与所有原生配体接合的能力。因此，我们试图探索单个和组合的Y152和Y199取代，并就其生化行为对其进行表征，目的是识别不能与任何天然配体结合的单突变和双突变变体。还良好表达和组装的那些变体是特别受关注的，因为这些表明了可以更容易产生用于分析的惰性配体。
经由短因子Xa识别性Ile-Glu-Gly-Arg接头(SEQ ID NO:38)，将天然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“工程化的NKG2D”或“eNKG2D”，作为融合体克隆至人类IgG1 Fc的C-末端(无Fab结构域)，并且可互换地称为Fc-NKG2D.wt或NKG2D.wt以及Fc-eNKG2D或eNKG2D(SEQ IDNO:40-58)。合成了对应于MHCI信号序列(SEQ ID NO:36和37)、具有接头的人类IgG1 Fc(SEQ ID NO:39)和NKG2D胞外域变体(SEQ ID NO:59-77)的
Figure BDA0003145238650000161
DNA片段(IntegratedDNA Technologies，San Diego，CA)，并将其插入到pD2610-V12(ATUM，Newark，CA)中。在Expi293TM细胞(ThermoFisher Scientific,Waltham,MA)中瞬时表达在Y152、Y199或Y152/Y199突变组合(图18)处探索取代的DNA构建体，并通过蛋白A亲和层析(产品编号20334，Pierce Biotechnology，Rockford，IL)纯化分泌的蛋白。通过尺寸排阻色谱(SEC)在AktaPur Superdex柱上表征洗脱的物质，并且将正确组装的尺寸合适的材料进行分馏并从聚集体峰中分离出来，然后再纳入测定。
纯化的NKG2D.Y199A-Fc融合体的SEC表征显示了主要聚集材料的组成(图2)。相比之下，天然Fc-NKG2D融合体和Fc-NKG2D.Y152A融合材料通过离散的非聚集峰区分开来，该峰容易与更快迁移的聚集体区分。Y199A突变对聚集的影响在Y152A/Y199A双突变体Fc-NKG2D融合变体中也是明显的，表明其对蛋白质错误折叠具有最重要的影响(图2)。因此，在NKG2D变体中包括Y199A与Y152突变的任何组合的这一方面对生产随后工程化工作所需的材料提出了挑战，并引起了关于在细胞表面上组装和呈递的担忧。因此，努力探索在Y152和Y199处的其它取代，它们可以组合产生更强的分子。将eNKG2D组合的Y152和Y199候选突变体作为Fc融合体进行检测并详述于(图18)。此外，通过SEC对所有纯化和表达的Fc-eNKG2D融合候选物进行作图，并且它们的色谱图揭示了不同水平的聚集体形成(图2和3，图18)。在第152位残基处探索的单个氨基酸取代中，丙氨酸、丝氨酸、苏氨酸和缬氨酸都不影响Fc-NKG2D分子的组装，但Y152-亮氨酸(Y152L)产生了高度聚集的材料。与丙氨酸相似，在第199位谷氨酸和天冬氨酸都不耐受，但苯丙氨酸仅适度地增加聚集体形成。在研究的突变组合中，Y152A/Y199F、Y152S/Y199F、Y152T/Y199F和Y152F/Y199F没有负面地影响所需的二聚体形成，而其它组合导致聚集增加(图18，图2和3)。
实施例2：产生具有非天然NKG2D配体变体的基于抗体的双特异性分子“MicAbody”
为了产生与人类IgG1融合的非天然MicA变体，使用引物PCR扩增编码例如MICwed(SEQ ID NO:79)和MIC25(SEQ ID NO:81)的α1-α2结构域的DNA多核苷酸，所述引物还引入编码用于与人类IgG1的C-末端κ轻链(SEQ ID NO:84)融合的APTSSSGGGGS接头或用于与C-末端重链(SEQ ID NO:82)融合的GGGS接头的多核苷酸。此外，将两个突变引入重链的CH2结构域中——D265A/N297A(Kabat编号；图13A和13B)，这降低了与所有FcγR受体的结合，从而消除了抗体依赖性细胞毒性(ADCC)功能(Shields et al.,2001JBC,276:6591-6604)。类似地克隆了编码野生型ULBP2(ULBP2.wt)的α1-α2结构域而无其GPI-连接的多核苷酸(SEQID NO:12)，并将其融合到编码接头和IgG1重链或轻链的DNA多核苷酸上。这些双特异性抗体——单数形式称为“MicAbodyTM”、复数形式称为“MicAbodies”——对于融合的α1-α2结构域是二价的。用于产生用于探索eNKG2D工程化的MicAbodies的抗体的实例包括但不限于曲妥珠单抗(SEQ ID NO:94和96)和利妥昔单抗(SEQ ID NO:98和100)，随后分别称为“曲妥珠单抗-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(New England Biolabs Inc.,Ipswich,MA)将融合构建体分别插入pD2610-V12(ATUM,Newark,CA)。对于识别特定抗原的给定抗体，将编码与天然或非天然NKG2D配体融合的重链的质粒和编码与天然或非天然NKG2D配体融合的轻链的质粒共转染，以在Expi293TM细胞(ThermoFisher Scientific,Waltham,MA)中瞬时表达。或者，将编码融合至天然或非天然NKG2D配体的重链的质粒和用于轻链的质粒共转染。通过蛋白A亲和层析(产品编号20334，Pierce Biotechnology，Rockford，IL)纯化分泌的双特异性抗体，通过尺寸排阻层析(SEC)在Akta Pur Superdex柱上表征洗脱的材料，并根据需要进行分级分离。此外，对纯化的样品进行SDS-PAGE分析以验证融合的重链和融合的轻链种类的预期分子量。
实施例3：识别经修饰的NK2GD变体，其不能结合天然NKG2D-结合配体或不能结合对野生型NKG2D具有增强结合性的非天然配体
使用基于培养板的ELISA方法，分析α1-α2变体对天然(野生型)NKG2D和非天然eNKG2D蛋白质的细胞外结构域的结合亲和力。在磷酸盐缓冲液(PBS)中使用1μg/mL的包被浓度，将SEC分级的天然Fc-NKG2D和非天然Fc-eNKG2D融合体中的每一种在4℃下包被在Nunc Maxisorp 96孔板(Thermo Fisher Scientific，Waltham，MA)的不同孔上过夜。在20-22℃下，在PBS/0.05％Tween-20(PBS-T)中将板洗涤三次，并在20-22℃下用在PBS中的0.5％牛血清白蛋白(PBS-B)封闭2小时。针对板结合的天然或非天然Fc-NKG2D融合体，在PBS/0.5％牛血清白蛋白(BSA)/0.05％Tween-20(PBS-BT)中，在20-22℃下滴定MicAbodies60分钟，在20-22℃下用PBS-T洗涤3次，并使用在PBS-BT中的HRP-缀合的抗人κ(Abcam，Cambridge MA)检测结合的双特异性蛋白，并用1-StepTM Ultra TMB ELISA底物溶液(Thermo Fisher Scientific，Waltham，MA)显影。ULBP2.wt利妥昔单抗-MicAbody(SEQ IDNO:98和106)的结合在野生型NKG2D和eNKG2D变体之间有区别，后者结合降低，并且配体变体-MICwed(SEQ ID NO:96和102)和MIC25(SEQ ID NO:96和104)-在识别消除配体结合的eNKG2D变体方面更严格。每种eNKG2D变体针对所有三种双特异性配体的结合行为揭示了NKG2D修饰的组合，该组合导致最大程度地降低了野生型和变体配体的结合，并使得能够选择先导惰性NKG2D变体。
使用FortéBio Octet系统(全为FortéBio LLC，Fremont，CA)，用生物层干涉测量法(BLI)也进行了eNKG2D变体与配体结合的其它生物物理分析。对于这些实验，人类NKG2D配体MICA-Fc、MICB-Fc、ULBP1-Fc、ULBP2-Fc、ULBP3-Fc和ULBP4-Fc购自R&D Systems,Inc.(Minneapolis，MN)。MicAbody形式的配体被捕获在抗人IgG Fc捕获(AHC)生物传感器尖端上。在建立基线后，将尖端暴露于在300nM至0.41nM范围内滴定系列的Fc-eNKG2D融合蛋白，并且用在PBS-BT中进行的所有步骤监测结合/解离动力学。随后，将Fc-eNKG2D融合蛋白捕获到AHC尖端上，并滴定MicAbodies以表征结合动力学。
为了测定由天然NKG2D与MICwed或MIC25结合所定义的最大响应，将天然Fc-NKG2D融合体捕获到AHC生物传感器上，并将20nM曲妥珠单抗-MICwed或20nM曲妥珠单抗-MIC25MicAbodies孵育两分钟，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，以Fc-NKG2D.wt建立的最大结合响应的百分比对每个eNKG2D的结合水平进行排名(图19)。对于MICwed，除Y199F外，所有单突变Fc-eNKG2D变体的响应减少至50％。Y199F保持了100％结合响应。然而，所有双突变Fc-eNKG2D变体完全消除了与MICwed的结合。对于MIC25，相对于野生型Fc-NKG2D结合，所有单突变Fc-eNKG2D变体和Y152V/Y199F保持了100％结合响应。然而，多种双突变Fc-eNKG2D变体，包括Y152A/Y199F、Y152S/Y199F和Y152T/Y199F，结合降低至50％。
使用Fc-eNKG2D融合体作为捕获剂的ELISA测定用ULBP2.wt、MICwed、MIC25MicAbodies进行，从300nM开始滴定(图4)。当可能时，使用GraphPad Prism计算EC50值(图20)。天然NKG2D结合基于ULBP2、MICwed和MIC25的MicAbody，计算的亲和力作为Kds值分别为1.4、0.007和0.005nM。尽管ULBP2和MICwed MicAbody与所有单突变eNKG2D候选物的亲和力降低，但保留了MIC25与eNKG2D候选物的结合性。然而，所有双突变eNKG2D候选物已消除或显著降低了与Micabody形式的所有三种配体-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)与基于ULBP2、MICwed和MIC25的MicAbody的结合降低或消失(图19和20)。此外，eNKG2D 5、7和8具有最小量的聚集，表明293T表达时更强的蛋白质组装(图18)。更仔细地检查了eNKG2D5(SEQ ID NO:48)与野生型配体的结合，如在Octet AHC尖端上捕获的MicAbodies。相对于天然(SEQ ID NO:40)NKG2D，单突变体Fc-NKG2D.Y152A(SEQ ID NO:41)与所有天然配体的结合降低(图5)。相对于Y152AeNKG2D，eNKG2D5(Y152A/Y199F)的结合的响应曲线甚至进一步降低。选择eNKG2D5(Y152A/Y199F，下文称为“AF”或“NKG2D.AF”)作为先导NKG2D变体，对其工程化同源选择性、正交、非天然的配体。
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
我们采用噬菌体展示来工程化正交的非天然α1-α2结构域，其表现出与NKG2D.AF(SEQ ID NO:48)受体的选择性结合。作为起点，选择对天然、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，用于进一步诱变和通过噬菌体展示筛选。针对ULBP2.R80W(SEQ ID NO:108)的α1-α2结构域产生合成的DNA文库，该结构域另外具有C8S突变以消除潜在的二硫键。使用NNK密码子替换配体上在结合态下位置与天然NKG2D受体上Y152和Y199位置紧密相邻的氨基酸残基的密码子；文库由位置154-159处的NNK密码子组成(图1B；SEQ ID NO:110)。将文库克隆为与M13噬菌体的pIII次要外壳蛋白的融合体，并根据标准方法(Andris-Widhopf,J.,Steinberger,P.,Fuller,R.,Rader,C.,and Barbas,C.F.,3rd.(2011))在SS320大肠杆菌细胞中产生展示诱变的α1-α2结构域变体的噬菌体颗粒。通过在非生物素化的天然Fc-NKG2D.wt竞争蛋白存在下，选择性捕获结合生物素化的Fc-NKG2D.AF蛋白的噬菌体克隆，对这些α1-α2噬菌体展示文库进行分选，以便对非天然NKG2D.AF受体具有高结合亲和力。通过使用增加浓度的非生物素化的天然Fc-NKG2D进行多轮竞争性选择，从而富集了选择性克隆。
四轮选择后，将噬菌体克隆分别排列在96孔格板中，进行spot ELISA以验证与板结合的非天然NKG2D.AF和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(ThermoFisher Scientific,Waltham,MA)、链霉亲和素-HRP检测(R&DSystems,Minneapolis,MN)和1-Step Ultra TMB ELISA显影(ThermoFisher Scientific,Waltham,MA)检测结合的噬菌体。每个克隆的spot ELISA信号表达为噬菌体结合NKG2D.AF与噬菌体结合NKG2D.wt的比率。对比率大于或等于14的那些噬菌体进行测序，以识别NNK诱变区内的特定突变。图21显示了选择性结合的NKG2D.AF的各α1-α2噬菌体变体的所选氨基酸残基。在识别代表相同序列的多个克隆的情况下，绘制ELISA信号的比率，并通过数据点的聚类验证噬菌体克隆的一致性(数据未显示)。
在单独的单一培养中扩增在ELISA中识别的三十种变体，以产生高滴度微批次的噬菌体。将纯化的噬菌体浓度归一化为OD268＝0.5，然后针对板结合的Fc-NKG2D.AF或Fc-NKG2D.wt进行1:3系列稀释，如上所述进行噬菌体检测和ELISA显影。以这种方式测定的所有三十种变体一致地证明了与NKG2D.AF的选择性结合，与NKG2D.wt结合很少至不结合(图6)，即使在测定的最高噬菌体浓度下亦是如此。所选噬菌体还显示两个或多个噬菌体浓度对数的偏移，以实现在NKG2D.AF和NKG2D.wt之间的半最大结合。
为了确认NKG2D.AF-选择性α1-α2结构域变体保留了在抗体融合中的特异性结合特性，将21种变体(图22；例如SEQ ID NO:111-118)作为与APTSSSGGGGS接头的C-末端融合克隆到利妥昔单抗抗体的轻链(SEQ ID NO:119-126)。将得到的融合体通过Gibson克隆(New England Biolabs Inc.,Ipswich,MA)克隆到哺乳动物表达载体pD2610-V12(ATUM，Newark，CA)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(ThermoFisher Scientific，Waltham，MA)中进行瞬时表达，并使用标准蛋白-A亲和层析(产品编号20334，Pierce Biotechnology，Rockford，IL)纯化。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，显示相对于天然NKG2D.wt，它们对NKG2D.AF具有显著更高的结合亲和力(图22)。总之，这些数据证明，本发明的非天然的正交α1-α2结构域对非天然NKG2D.AF受体具有高亲和力结合，并且与天然NKG2D受体的结合亲和力显著降低。此外，这些正交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，并且例如在嵌合抗原受体(CAR)T细胞的背景下用于将非天然NKG2D.AF受体重新导向特异性抗原。
实施例5:识别非天然NKG2D配体，其可通过选择性结合一种或另一种区分非天然NKG2D受体变体
如上所述，使用非天然ULBP2.R80wα1-α2结构域(SEQ ID NO:108)作为起始点，进行了噬菌体展示，以工程化对NKG2D.Y152A(此后称为NKG2D.YA)受体具有选择性结合的正交非天然α1-α2结构域。在非生物素化的天然Fc-NKG2D.wt(SEQ ID NO:40)竞争蛋白存在下，通过选择性捕获结合到生物素化的Fc-NKG2D.YA(SEQ ID NO:41)蛋白的噬菌体克隆，对α1-α2噬菌体展示文库进行淘选，以获得对非天然Fc-NKG2D.YA受体的高结合亲和力。附加的噬菌体克隆验证工作导致识别了与Fc-NKG2D.wt相比与Fc-NKG2D.YA优先结合的变体(图23)。例如，通过ELISA和Octet分析(都以单体His-标记的和双特异性抗体融合的形式)一致地证明，相对于天然NKG2D.wt，ULBP2.S3(SEQ ID NO:127)与非天然NKG2D.YA选择性结合。这代表了与非天然NKG2d受体具有高亲和力结合的正交α1-α2结构域的本发明的一个独特形式(在这种情况下，NKG2D.YA与示例4中的NKG2D.AF相反)。另外，正交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NKG2D受体重新导向由融合的异源肽(如抗体)确定的特异性分子。
为了确定与NKG2D.YA(ULBP2.S3,SEQ ID NO:127)选择性结合的非天然α1-α2结构域和与NKG2D.AF选择性结合的非天然α1-α2结构域是否可区分这两种非天然受体变体，进行ELISA滴定。直接比较所有21种所选的结合NKG2D.AF的α1-α2变体与NKG2D.AF和NKG2D.YA的结合。其中，四种表明了不能结合NKG2D.wt、对NKG2D.AF具有强亲和力的特性，并且相对于NKG2D.AF，大大降低(15-20倍)或消除了与NKG2D.YA的结合(图7)。对于这四种非天然ULBP2α1-α2变体-ULBP2.C、ULBP2.R、ULBP2.AA和ULBP2.AB(SEQ ID NO:111,113,115和117)，还使用NetMHC4.0服务器(对于肽-MHC I类结合，用9-聚体肽分析查询所有HLA超型代表；http://www.cbs.dtu.dk/services/NetMHC/)和NetMHCII 2.3服务器(对于肽-MHC II类结合，用15-聚体肽分析查询HLA-DR、HLA-DQ、HLA-DP单倍型；http://www.cbs.dtu.dk/services/NetMHCII/)，相对于野生型ULBP2肽序列(SEQ ID NO:4)检查了预测的免疫原性谱的改变，前述两种算法都是由丹麦技术大学开发的(http://www.bioinformatics.dtu.dk/；Andreatta M and Nielsen M,Gapped sequencealignment using artificial neural networks:application to the MHC class Isystem,2016Bioinformatics,32:511,PMID:26515819；Jensen KK,Andreatta M,Marcatili P,Buus S,Greenbaum JA,Yan Z,Sette A,Peters B,and Nielsen M,Improvedmethods for predicting peptide binding affinity to MHC class I molecules,2018Immunology,PMID:29315598)。向ULBP2.C、ULBP2.R和ULBP2.AB中并入突变不增加预测的免疫原性，而对于少数单倍型，ULPB2.AA略微增加(图8和9)。由于ULBP2.R对NKG2D.AF的特异性及其缺乏可预测的免疫原性，选择ULBP2.R用于进一步的ELISA分析，以直接比较其与ULBP2.S3(NKG2D.YA-选择的非天然正交配体)、ULBP2.R80W(对野生型NKG2D具有增强亲和力的非天然配体)和野生型ULBP2(ULBP2.wt)的结合行为。针对野生型NKG2D(NKG2D.wt)和两种惰性非天然变体NKG2D.YA和NKG2D.AF，测定了四种利妥昔单抗-MicAbody试剂(SEQID 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10)。数据表明，NKG2D.YA-选择的变体ULBP2.S3为一种以高亲和力与NKG2D.YA结合的MicAbody，但不接合NKG2D.AF或天然NKG2D。此外，NKG2D.AF-选择的变体ULBP2.R以MicAbody形式以高亲和力结合NKG2D.AF，但不接合NKG2D.YA或天然NKG2D。这些结果证明，探索NKG2D-MIC配体轴和开发独特成对的新型选择性非天然NKG2D受体及它们各自的同源非天然MIC配体结合配偶体具有巨大的潜力。
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至异源靶向多肽的正交α1-α2结构域控制
强烈寻求选择性控制CAR-T细胞疗法的方法，以减轻毒性和改善抗肿瘤功效(Gilland June,前面所引用的)。先前已经尝试使用CD16的胞外域开发CAR，然后其可以通过治疗性单克隆抗体的Fc结构域接合，允许基于抗体的CAR-T靶向控制(Chang et al.,前面所引用的)。然而，基于CD16的CAR-T细胞可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会遇到来自NK细胞、PMN、单核细胞和巨噬细胞上的内源性CD16受体的竞争。这两个特征均分别造成肿瘤外毒性和不良药物动力学的问题。
天然NKG2D配体存在于某些健康组织和许多应激组织上，使用目前的NKG2D CAR方法(VanSeggelen et al.2015)产生了极高的毒性风险。Y152A非天然NKG2D受体特异性结合非天然α1-α2结构域NKG2D配体，构成了一种手段的实例，通过该手段，使用包含所发明的NKG2D配体的非天然α1-α2结构域的双特异性蛋白质，可选择性控制非天然NKG2D CAR的活性。
我们用包含NKG2D的经修饰的Y152A/Y199F(“AF”)胞外域的受体工程化CAR-T细胞，所述胞外域缺乏与所有天然NKG2D配体的结合，或缺乏与Y152A修饰的NKG2D(NKG2D.YA)正交并同源的先前描述的非天然α1-α2结构域。所发明的同源非天然α1-α2结构域以高亲和力与非天然NKG2D.AF胞外域结合并避免与天然NKG2D胞外域和与NKG2D.YA胞外域结合。因此，工程化的α1-α2结构域表现出相对于天然NKG2D和非天然NKG2D.YA，对非天然NKG2D.AF胞外域具有强选择性，其代表了一种理想的系统，可用于选择性控制非天然NKG2D CAR受体，或可以被本发明的非天然α1-α2结构域选择性接合的融合到非天然NKG2D胞外域的任何受体或蛋白质。本发明进一步使得单细胞表达两种不同的CAR——一种包含NKG2D.YA且另一种包含NKG2D.AF——每种使用明显不同的胞内结构域进行信号传导。这些不同的CAR通过细胞外暴露于各自的同源正交MicAbody或另一种非抗体融合多肽而具有对细胞活性的独立双重控制。
为了证明用使用非天然NKG2D.AF胞外域的嵌合受体构建的CAR-T细胞的选择性控制，我们基于先前的工作，使用4-1BB/CD3-ζCAR构建体(Campana专利8,399,645)，将各自的NKG2D胞外域融合到CAR的CD8铰链区(SEQ ID NO:151，153，155)，构建了具有NKG2D.wt(SEQID NO:135)、非天然NKG2D.YA(SEQ ID NO:137)或非天然NKG2D.AF(SEQ ID NO: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类CD8-阳性T细胞中表达。HeLa细胞在其表面具有组成性上调水平的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；人类ULBP-2/5/6抗体，R&D Systems，Minneapolis，MN)。转染HeLa细胞以在表面上过表达天然ULBP1或NKG2D.AF-选择的变体ULBP2.R的细胞，并且这些细胞用作体外杀伤测定的靶标。HeLa靶细胞预先负载钙黄绿素并在不断增加的效应子与靶标(E:T)比率下，暴露于NKG2D.wt-CAR、NKG2D.YA-CAR或NKG2D.AF-CAR CD8细胞中五小时，之后对释放到上清液中的钙黄绿素量进行定量并且归一化至在洗涤剂处理后释放的总钙黄绿素(图11)。由于天然表达在HeLa细胞表面上的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细胞接合，并实现细胞溶解。然而，即使在高E:T比率下，NKG2D.YA-和NKG2D.AF-CAR转导的CD8细胞都显示非常少的天然HeLa细胞裂解，与未转导的CD8T细胞的活性水平相当。当ULBP1在HeLa细胞表面过表达时，仅NKG2D.wt-CAR CD8T细胞显著裂解它们。在高E:T比率下，使用NKG2D.YA-CAR细胞存在一些额外的杀伤，但这在NKG2D.AF-CAR细胞中是不存在的，表明双突变Y152A/Y199F使得NKG2D比单一Y152A突变更惰性。在过表达NKG2D.AF-选择性非天然ULBP2.R的HeLa细胞中，NKG2d.wt-CAR细胞导向裂解(由于内源MIC配体的识别)，而NKG2D.AF-CAR细胞导向显着水平的裂解，与受体与其选择性配体的接合一致。
为了证明NKG2D.YA-或NKG2D.AF-CAR细胞的裂解只能由适当的同源靶向MicAbody来引起，将Ramos细胞与与非天然ULBP2.S3或ULBP2.R正交配体连接的基于利妥昔单抗的MicAbodies一起用作细胞溶解的靶标。如图12A所示，利妥昔单抗-ULBP2.S3 MicAbody可引起NKG2D.YA-CAR CD8细胞的细胞杀伤活性，但不是NKG2D.AF-CAR细胞，而利妥昔单抗-ULBP2.R MicAbody可以引起NKG2D.AF-CAR细胞的活性，而不是NKG2D.YA-CAR细胞。这进一步证明了两种非天然ULBP2变体对其同源非天然NKG2D变体的选择性，其中将它们工程化为优选配偶体。为了证明MicAbody抗体部分的特异性，使用通过利妥昔单抗-ULBP2.R、曲妥珠单抗-ULPB2.R(SEQ ID NO:95和133，分别为重链和轻链)或两者的等摩尔组合在MicAbody的饱和总浓度下孵育预处理的NKG2D.AF-CAR CD8 T-细胞，进行了体外杀伤测定。通过洗涤除去未结合的MicAbody后，将CD8细胞应用于已经预先加载钙黄绿素的Ramos细胞(表达CD20，利妥昔单抗的靶标)或CT26-Her2(经转染以表达人类Her2的小鼠细胞系)。在两种不同的E:T比率下孵育两小时后，定量所释放的钙黄绿素的量。如图12B所示，当使用利妥昔单抗-MicAbody预处理细胞时，只有Ramos细胞被裂解，而曲妥珠单抗-MicAbody仅针对CT26-Her2细胞引起细胞裂解活性。然而，当同时使用利妥昔单抗-和曲妥珠单抗-ULBP2.RMicAbodies预处理NKG2D.AF-CAR CD8细胞时，两个靶细胞系都被裂解，证明这些CAR细胞——由于在受体和配体之间工程化的选择性优先配对——容易被复用，从而被引导同时接合不同的肿瘤靶。
实施例7:正交α1-α2结构域作为选择性地将细胞因子递送至表达非天然工程化的NKG2D的T细胞的手段
双特异性MicAbodies(图13B)利用抗体的抗原靶向Fv结构域以及正交α1-α2结构域与工程化的非天然NKG2D.YA或NKG2D.AF(分别为SEQ ID NO:137和139)之间的优先相互作用，可有效引起携带NKG2D.YA-或NKG2D.AF-CAR的T细胞(分别为SEQ ID NO:153和155)的细胞溶解能力，从而消除表达抗原的靶细胞。另外，可利用同源正交配体和非天然的、经修饰的(也称为“工程化的”)NKG2D(eNKG2D)之间的高度选择性相互作用，以选择性地将分子递送至携带eNKG2D-胞外域(eNKG2D-ecd)的细胞。如果与正交配体融合的异源原子或异源分子(载荷/载物)在递送至非-eNKG2D表达细胞时具有潜在不良功能的固有生物活性，则可以探寻减少与原生受体或靶标的相互作用的生物活性分子中的突变。因此，当与正交配体融合时，最终分子对所有生物功能都是有效惰性的，除非存在具有同源eNKG2D受体的细胞。只有当正交配体和eNKG2D受体之间发生高亲和力相互作用时，生物活性分子的亲和力降低的突变体和其天然靶标或受体之间的相互作用才被促进，并且生物活性分子的残留功能才得以实现。为了将实体定向到表达eNKG2D的细胞而与所述实体融合的所有正交配体融合统称为MicAdaptor，并且原则上可以采取直接蛋白质融合的形式，作为有效负载与正交配体之间的单一多肽，其在N-或C-末端具有或不具有用于测定或纯化的接头或标签(例如His标签)(图13C)。另外，如果需要增强的血清稳定性，则可以仅作为CH2-CH3结构域或者以完整抗体的形式包括抗体Fc结构域，这取决于所需的独特有效负载的数目、正交配体或异源载物的化合价、以及促进所有组分的最大功能的一般实验确定的架构(图13D)。
为了确定细胞因子是否可以选择性地递送至NKG2D.YA-CAR表达细胞(SEQ ID NO:153)，表达了同源正交配体ULBP2.S3(U2S3，SEQ ID NO:127)，融合至Fc1的N-末端，该Fc1已在两个残基处被改变以在Fc均二聚化的界面上表达带负电荷的天冬氨酸残基(SEQ ID NO:189)。将突变形式的人类IL2(mutIL2)(含有显著降低细胞因子对IL2R-α复合物的亲和力的两个突变R38A/F42K)(K.M.Heaton,G.Ju,and E.A.Grimm,Human Interleukin 2AnaloguesThat Preferentially Bind the Intermediate-Affinity Interleukin 2Receptor Leadto Reduced Secondary Cytokine Secretion:Implications for the Use of TheseInterleukin 2Analogues in Cancer Immunotherapy,1993Cancer Res 53:2597,PMID:8495422；K.Sauvéet al.,Localization in Human Interleukin 2of the Binding Siteto the Alpha Chain(P55)of the Interleukin 2Receptor,1991PNAS 88:4636,PMID:2052547)，融合到在两个残基处被改变以在Fc均二聚化的界面上表达带正电荷的赖氨酸残基的Fc2的C-末端(SEQ ID NO:183)。将两者独立地克隆到哺乳动物表达载体pD2610-V12(ATUM，Newark，CA)中，根据制造商的方案共转染到Expi293TM细胞(ThermoFisherScientific，Waltham，MA)中，并使用标准蛋白-A亲和层析(产品编号20334，PierceBiotechnology，Rockford，IL)纯化。通过尺寸排阻色谱(SEC)在Akta Pur Superdex柱上将纯化的物质分级分离。Fc1上带负电荷的残基和Fc2上带正电荷的残基提供了静电转向效应(Kannan Gunasekaran et al.,Enhancing Antibody Fc Heterodimer Formationthrough Electrostatic Steering Effects:Applications to Bispecific Moleculesand Monovalent IgG,2010J Biol Chem 285:19637,PMID:20400508)，其促进对于正交U2S3配体和mutIL2为单价的分子的异二聚体组装(图13Da)。另外，研究了包含ULBP2正交配体与细胞因子融合体的其它组合的MicAdaptor——作为异二聚体Fc1/Fc2融合体或作为其中两种组分直接融合的单一多肽——并且如图24中详述和如上所述类似地克隆、共表达和纯化它们的DNA。根据需要，在测定中，包括重组人类IL2(rhIL2，Peprotech)和重组人类IL15(rhIL15，Peprotech)作为对照。
转导CD8人类T细胞以表达NKG2D.wt-CAR构建体(SEQ ID NO:151)或NKG2D.YA-CAR构建体(SEQ ID NO:153)并暴露于30IUe/mL对照细胞因子或各种MicAdaptor三天，并且使用WST-1细胞增殖试剂(Millipore Sigma)量化细胞增殖水平。对照rhIL2促进了两种CAR-表达细胞的增殖，而单独的mutIL2则不能，如从降低的与IL2R-α的结合能力以及因此降低的通过IL2R-β/γ-C发出信号的能力所预期的一样(图15A)。当MicAdaptor包含非选择性U2R80W配体(U2R80W-mutIL2，SEQ ID NO:177)时，其以高亲和力与野生型和经修饰的NKG2D.YA受体接合，表达任一CAR的细胞通过增殖响应。然而，当mutIL2与正交配体(诸如仅选择性接合NKG2D.YA的ULBP2.S2(SEQ ID NO:179)、MicAdaptor、仅与NKG2D.YA接合的U2S2-mutIL2)融合时，仅NKG2D.YA-CAR细胞通过增殖响应。当融合到U2S2的细胞因子是具有V49D突变的IL15的突变形式时(Bernard,J.et al.,Identification of anInterleukin-15αReceptor-binding Site on Human Interleukin-15,2004Journal ofBiological Chemistry,279:24313,PMID:15039446)时，获得了类似的结果，所述V49D突变减少了与IL15R-α的接合(图15A)。当NKG2D.YA-CAR-T细胞与这些试剂共培养七天时，％GFP+(指示与未转导的GFP-阴性细胞相比存在的CAR-表达细胞的百分比)增加，但仅当存在正交ULBP2.S2配体(U2S2-mutIL2或U2S2-Fc1/Fc2-mutIL2，SEQ ID NO见图24)且不存在非选择性U2R80W变体(其结合在人类CD8细胞上组成型存在的天然野生型NKG2D受体)时如此(图15B)。IL21也被作为野生型融合(IL21.wt)进行了研究，具有影响IL21R-α结合的单突变(D18A或E109R)，或具有包括所述两个突变的版本(D18A/E109R)(Kang,L.et al.,RationalDesign of Interleukin-21Antagonist through Selective Elimination of theγCBinding Epitope,2010Journal of Biological Chemistry,285:12223,PMID:20167599)。比较未转导的CD8细胞或NKG2D.YA-CAR表达细胞，仅携带NKG2D.YA的细胞对所有版本的IL21融合体(SEQ ID NO见图24)，包括对IL21.wt融合体，作出响应(图15C)。有趣的是，尽管与无细胞因子对照相比，未转导的和NKG2D.YA-CAR细胞均响应rhIL2而扩增，但NKG2D.YA-CAR细胞的增殖量更高。NKG2D.YA-CAR表达细胞的增殖响应不依赖于NKG2D.YA结构域的一般化结合或接合，因为U2S3-Fc1/Fc2(具有单一U2S3结构域且没有附着的细胞因子或细胞因子突变体的异二聚体Fc分子)和利妥昔单抗-MicAbody(对于U2S3结构域是二价的，但缺乏任何细胞因子组分，图13Bb)在三天的培养过程中不诱导增殖，甚至在比rhIL2对照高得多的IUe/mL浓度下亦如此(图16C)。
实施例8:顺式或反式细胞内共刺激结构域的存在促进了携带非天然的、经修饰的NKG2D的细胞对细胞因子和细胞因子MicAdaptor的响应
在证明了在嵌合抗原受体构建体的背景下，经修饰的NKG2D.YA结构域实际上确实充当了用于递送与正交配体附着的异源载物的高度选择性对接位点后，我们试图确定NKG2D.YA受体是否不仅对于靶向递送细胞因子是必需的，而且对于靶向递送细胞因子也是足够的，所述细胞因子然后可以作用于接受细胞。NKG2D.YA胞外结构域(NKG2D.YA-ecd)表达为除去了所有胞内组分的跨膜结构域，但保留了胞内eGFP标签(图14，SEQ ID NO:157)。转导CD8细胞以表达该“沉默CAR”，并证明在利妥昔单抗-ULBP2.S3 MicAbody(SEQ ID NO:98和129)存在下不能直接杀伤Ramos靶细胞，正如没有共刺激结构域所预期的(图16A)。重要的是，表达这种完全惰性或沉默CAR的细胞在暴露于U2S3-Fc1/Fc2-mutIL2(SEQ ID NO:189和193)时不增殖，但在与未转导的细胞相当的水平上响应(图16B)。这一观察结果——除了(a)与未转导的细胞相比，一致观察到rhIL2可使NKG2D.YA-CAR增殖水平更高(图15C、16B、16C)和(b)观察到仅NKG2D.YA-CAR细胞对包括IL21.wt在内的所有形式的IL21-MicAdaptor作出响应之外——导致我们推测NKG2D.YA-CAR构建体中存在的细胞内结构域增强了对这些细胞因子和细胞因子MicAdaptor的响应性。
为了检测这一点，产生了一系列CAR构建体，其中CAR的胞内结构域的信号基序被突变——4-1BB的两个TRAF2共有结合位点(SEQ ID NO:161)、CD3-ζ中的三对ITAM基序(SEQID NO:163)或组合的4-1BB/CD3-ζ突变体(SEQ ID NO:165)。将这些构建体(图14)转导到CD8细胞中，与所示细胞因子试剂共孵育，三天后量化增殖(图17A)。在所有测试条件下，NKG2D.YA-BB-CD3ΔITAM-GFP(SEQ ID NO:163)保留了与NKG2D.YA-CAR相当的增殖响应，由此证明CD3-ζ结构域(SEQ ID NO:145)对于在CAR背景下对细胞因子和细胞因子-MicAdaptor的响应性不是必需的。然而，NKG2D.YA-BBΔTRAF2-CD3ζ-GFP受体-表达细胞(SEQ ID NO:161)对所有细胞因子和细胞因子-MicAdaptor的响应性显著降低，在两个细胞内结构域携带突变的NKG2D.YA-BBΔTRAF2-CD3ΔITAM-GFP(SEQ ID NO:165)受体也是如此。这表明共刺激4-1BB(SEQ ID NO:143)在NKG2D.YA-CAR细胞针对细胞因子和细胞因子-MicAdaptor的响应中起作用，并且在细胞表面上表达的沉默CAR需要还具有细胞内共刺激结构域存在，以促进细胞因子-MicAdaptor响应性。NKG2D.YA-BB沉默CAR(SEQ ID NO:169,图14)不能引起MicAbody介导的细胞杀伤(图17B)，证明缺乏CD3-ζ的这种CAR架构适合作为用于MicAdaptor的沉默对接位点，并且使细胞因子-MicAdaptor信号传导成为可能，但不能起到介导MicAbody靶向细胞的细胞溶解。
为了进一步探究4-1BB结构域如何促成细胞因子和细胞因子-MicAdaptor响应性，生成了构建体，其中CD19scFv-CAR(基于FMC63 Fv)含有功能性4-1BB和CD3-ζ结构域(SEQID NO:173)的完整互补序列。将CD19scFv-CAR与NKG2D.YA-ecd共表达(图14)。两种组分都表达为具有T2A自切割肽基序的单一多肽，该T2A自切割肽基序将上游CD19scFv-CAR构建体与下游NKG2D.YA-ecd分开，其具有独立的GMSCFRα链信号序列、CD8a铰链和CD8a跨膜结构域。将该构建体转导入CD8T细胞中，并且通过流式细胞术检查GFP信号和藻红蛋白缀合的MicAbody染色，分别验证了表面上CD19scFv-CAR和NKG2D.YA-ecd的共表达。将这些细胞与细胞因子和细胞因子-MicAdaptor一起孵育三天，并通过WST测定量化增殖。NKG2D.YA-CAR对rhIL2和U2S3-Fc1/Fc2-mutIL2均产生了预期的响应，尽管仅携带CD19scFv-CAR(SEQ IDNO:171)的对照细胞随rhIL2扩增，并且以较低程度扩增至U2S3-Fc1/Fc2-mutIL2的最高浓度(300IUe/mL)(图17C)。与仅表达CD19scFv-CAR(SEQ ID NO:171)的细胞相比，CD19scFv-CAR表达细胞(SEQ ID NO:173)上NKG2D.YA-ecd的共表达表现出对U2S3-Fc1/Fc2-mutIL2细胞因子-MicAdaptor更强的增殖响应。在此背景下，4-1BB结构域以反式组成型方式提供给NKG2D.YA-ecd。这些数据证明，顺式或反式4-1BB结构域促进了NKG2D.YA-ecd表达细胞对细胞因子和细胞因子-MicAdaptor的响应，并且NKG2D.YA-ecd结构域可以与共刺激性含4-1BB的CAR细胞共表达，以向过继性细胞治疗策略的工程化细胞提供增加的通用功能。这种功能性不仅限于在配体/沉默CAR接合后接合表面受体，而且可以通过并入胞质序列基序而扩展至细胞内递送(K.N.Pandey,Functional roles of short sequence motifs in theendocytosis of membrane receptors,2009Front Biosci,14:5339,PMID:19482617)，所述胞质序列基序促进非天然NKG2D变体的周转，使得任何结合的MicAdaptor都被共内在化并且在细胞内递送异源载物。
序列表
<110> 修普霍斯生物科学有限公司（XYPHOS BIOSCIENCES INC.）
<120> 不直接向其附着的细胞传导信号的非天然NKG2D受体
<130> A251193
<150> US 62/755,776
<151> 2018-11-05
<160> 202
<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> 合成肽人类IgG1 Fc与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，编码人类IgG1 Fc与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> 合成肽人类IgG1 Fc-NKG2D融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152A融合
<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> 合成肽人类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> 合成肽人类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> 合成肽人类IgG1 Fc-NKG2D Y199F eNKG2D1融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152S eNKG2D2融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152T eNKG2D3融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152V eNKG2D4融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152A/Y199F eNKG2D5融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152L/Y199F eNKG2D6融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152S/Y199F eNKG2D7融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152T/Y199F eNKG2D8融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152V/Y199F eNKG2D9融合
<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> 合成肽人类IgG1 Fc-NKG2D Y199D eNKG2D10融合
<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> 合成肽人类IgG1 Fc-NKG2D Y199E eNKG2D11融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152D/Y199D eNKG2D12融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152E/Y199E eNKG2D13融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152L eNKG2D14融合
<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> 合成肽人类IgG1 Fc-NKG2D Y152F/Y199F eNKG2D15融合
<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，编码人类IgG1 Fc-NKG2D融合
<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，编码人类IgG1 Fc-NKG2D Y152A
融合
<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，编码人类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，编码人类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，编码人类IgG1 Fc-NKG2D Y199F
eNKG2D1融合
<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，编码人类IgG1 Fc-NKG2D Y152S
eNKG2D2融合
<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，编码人类IgG1 Fc-NKG2D Y152T
eNKG2D3融合
<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，编码人类IgG1 Fc-NKG2D Y152V
eNKG2D4融合
<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，编码人类IgG1 Fc-NKG2D
Y152A/Y199F eNKG2D5融合
<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，编码人类IgG1 Fc-NKG2D
Y152L/Y199F eNKG2D6融合
<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，编码人类IgG1 Fc-NKG2D
Y152S/Y199F eNKG2D7融合
<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，编码人类IgG1 Fc-NKG2D
Y152T/Y199F eNKG2D8融合
<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，编码人类IgG1 Fc-NKG2D
Y152V/Y199F eNKG2D9融合
<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，编码人类IgG1 Fc-NKG2D Y199D
eNKG2D10融合
<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，编码人类IgG1 Fc-NKG2D Y199E
eNKG2D11融合
<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，编码人类IgG1 Fc-NKG2D
Y152D/Y199D eNKG2D12融合
<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，编码人类IgG1 Fc-NKG2D
Y152E/Y199E eNKG2D13融合
<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，编码人类IgG1 Fc-NKG2D Y152L
eNKG2D14融合
<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，编码人类IgG1 Fc-NKG2D
Y152F/Y199F eNKG2D15融合
<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> 合成肽人类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> 人类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> 合成肽人类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，编码人类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为a, c, g或t
<220>
<221> misc_feature
<222> (460)..(461)
<223> n为a, c, g或t
<220>
<221> misc_feature
<222> (463)..(464)
<223> n为a, c, g或t
<220>
<221> misc_feature
<222> (466)..(467)
<223> n为a, c, g或t
<220>
<221> misc_feature
<222> (469)..(470)
<223> n为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
<210> 157
<211> 473
<212> PRT
<213> 人工序列
<220>
<223> 合成肽NKG2D.YA_CD8铰链TM_EGFP沉默嵌合抗原受体
<400> 157
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 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> 158
<211> 1419
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码NKG2D.YA_CD8铰链TM_EGFP 沉默嵌合抗原受体
<400> 158
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 actgctcagg 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> 159
<211> 225
<212> PRT
<213> 人工序列
<220>
<223> 合成肽NKG2D.YA_CD8铰链TM沉默嵌合抗原受体
<400> 159
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
225
<210> 160
<211> 675
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码NKG2D.YA_CD8铰链TM沉默嵌合抗原受体
<400> 160
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 actgc 675
<210> 161
<211> 629
<212> PRT
<213> 人工序列
<220>
<223> 合成肽NKG2D.YA_CD8铰链TM_4-1BBtraf2_CD3ζ_EGFP
完全嵌合抗原受体
<400> 161
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 Ala Ala Ala Gly Cys Ser
245 250 255
Cys Arg Phe Pro Glu Ala Ala Ala 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> 162
<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_4-1BBtraf2_CD3ζ_EGFP完全嵌合抗原受体
<400> 162
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 gccgctgcag gctgctcctg cagattcccc 780
gaggctgccg caggcggctg 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> 163
<211> 629
<212> PRT
<213> 人工序列
<220>
<223> 合成肽NKG2D.YA_CD8铰链TM_4-1BB_CD3itam_EGFP完全嵌合抗原受体
<400> 163
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 Phe Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Phe 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 Phe Asn Glu Leu Gln Lys Asp Lys Met
325 330 335
Ala Glu Ala Phe Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
340 345 350
Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser Thr Ala Thr Lys Asp
355 360 365
Thr Phe 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> 164
<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_CD3itam_EGFP完全嵌合抗原受体
<400> 164
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 ccagctgttc aacgagctga acctggggag aagagaagag 900
ttcgacgtgc tggacaagcg gagaggcaga gatcctgaga tgggcggcaa gcccagacgg 960
aagaatcctc aagagggcct gtttaatgag ctgcagaaag acaagatggc cgaggccttc 1020
agcgagatcg gaatgaaggg cgagcgcaga agaggcaagg gacacgatgg actgttccag 1080
ggcctgagca ccgccaccaa ggataccttt 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> 165
<211> 629
<212> PRT
<213> 人工序列
<220>
<223> 合成肽NKG2D.YA_CD8铰链TM_4-1BBtraf2_CD3itam_EGFP
完全嵌合抗原受体
<400> 165
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 Ala Ala Ala Gly Cys Ser
245 250 255
Cys Arg Phe Pro Glu Ala Ala Ala 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 Phe Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Phe 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 Phe Asn Glu Leu Gln Lys Asp Lys Met
325 330 335
Ala Glu Ala Phe Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
340 345 350
Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser Thr Ala Thr Lys Asp
355 360 365
Thr Phe 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> 166
<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_4-1BBtraf2_CD3itam_EGFP完全嵌合抗原受体
<400> 166
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 gccgctgcag gctgctcctg cagattcccc 780
gaggctgccg caggcggctg cgagctgaga gtgaagttca gccgttctgc cgacgctccc 840
gcctataagc agggacagaa ccagctgttc aacgagctga acctggggag aagagaagag 900
ttcgacgtgc tggacaagcg gagaggcaga gatcctgaga tgggcggcaa gcccagacgg 960
aagaatcctc aagagggcct gtttaatgag ctgcagaaag acaagatggc cgaggccttc 1020
agcgagatcg gaatgaaggg cgagcgcaga agaggcaagg gacacgatgg actgttccag 1080
ggcctgagca ccgccaccaa ggataccttt 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> 167
<211> 517
<212> PRT
<213> 人工序列
<220>
<223> 合成肽NKG2D.YA_CD8铰链TM_4-1BB_EGFP完全嵌合抗原受体
<400> 167
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 Ser Gly Ser
260 265 270
Gly Ser Gly Ser Gly Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr
275 280 285
Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His
290 295 300
Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys
305 310 315 320
Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp
325 330 335
Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg
340 345 350
Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro
355 360 365
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn
370 375 380
Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn
385 390 395 400
Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu
405 410 415
Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met
420 425 430
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
435 440 445
Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
450 455 460
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
465 470 475 480
Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
485 490 495
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
500 505 510
Asp Glu Leu Tyr Lys
515
<210> 168
<211> 1551
<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_EGFP
完全嵌合抗原受体
<400> 168
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 cgagctgtca ggctctggtt ctggcagcgg cagcatggtg 840
tctaaaggcg aggaactgtt caccggcgtg gtgcccattc tggtggaact ggacggggat 900
gtgaacggcc acaagtttag cgttagcggc gaaggcgaag gggatgccac atacggaaag 960
ctgaccctga agttcatctg caccaccggc aagctgcctg tgccttggcc tacactggtc 1020
accacactga catacggcgt gcagtgcttt agcagatacc ccgaccatat gaagcagcac 1080
gacttcttca agtccgccat gcctgagggc tacgtgcaag agcggaccat cttctttaag 1140
gacgacggca actacaagac cagggccgaa gtgaagtttg agggcgacac cctggtcaac 1200
cggatcgagc tgaagggcat cgacttcaaa gaggatggca acatcctggg ccacaagctc 1260
gagtacaact acaacagcca caacgtgtac atcatggccg acaagcagaa gaacggcatc 1320
aaggccaact tcaagatccg gcacaacatc gaggacggca gcgttcagct ggccgatcac 1380
taccagcaga acacccctat cggagatggc cctgtgctgc tccccgacaa tcactacctg 1440
agcacacaga gcgccctgag caaggacccc aacgagaaga gggatcacat ggtgctgctg 1500
gaatttgtga ccgccgcagg catcaccctc ggcatggacg aactgtacaa a 1551
<210> 169
<211> 269
<212> PRT
<213> 人工序列
<220>
<223> 合成肽NKG2D.YA_CD8铰链TM_4-1BB完全嵌合抗原受体
<400> 169
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
260 265
<210> 170
<211> 807
<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
完全嵌合抗原受体
<400> 170
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 cgagctg 807
<210> 171
<211> 736
<212> PRT
<213> 人工序列
<220>
<223> 合成肽CD19scFv_CD8铰链TM_4-1BB_CD3ζ_EGFP完全嵌合抗原受体
<400> 171
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 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
130 135 140
Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser
145 150 155 160
Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly
165 170 175
Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly
180 185 190
Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser
195 200 205
Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys
210 215 220
Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys
225 230 235 240
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly
245 250 255
Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro
260 265 270
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
275 280 285
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
290 295 300
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
305 310 315 320
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Ser Leu Lys Arg
325 330 335
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
340 345 350
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
355 360 365
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser Gly Ser Gly Ser Gly
485 490 495
Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile
500 505 510
Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser
515 520 525
Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe
530 535 540
Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
545 550 555 560
Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met
565 570 575
Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
580 585 590
Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
595 600 605
Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
610 615 620
Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
625 630 635 640
Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys
645 650 655
Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly
660 665 670
Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
675 680 685
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
690 695 700
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
705 710 715 720
Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
725 730 735
<210> 172
<211> 2208
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码
CD19scFv_CD8铰链TM_4-1BB_CD3ζ_EGFP完全嵌合抗原受体
<400> 172
atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60
cctgacatcc agatgaccca gacaaccagc agcctgtctg ccagcctggg cgatagagtg 120
accatcagct gtagagccag ccaggacatc agcaagtacc tgaactggta tcagcagaaa 180
cccgacggca ccgtgaagct gctgatctac cacaccagca gactgcacag cggcgtgcca 240
agcagatttt ctggcagcgg ctctggcacc gactacagcc tgacaatcag caacctggaa 300
caagaggata tcgctaccta cttctgccag caaggcaaca ccctgcctta cacctttggc 360
ggaggcacca agctggaaat cacaggcggc ggaggaagcg gaggcggagg atctggtggt 420
ggtggatctg aagtgaaact gcaagagtct ggccctggcc tggtggcccc atctcaatct 480
ctgagcgtga cctgtaccgt cagcggagtg tccctgcctg attatggcgt gtcctggatc 540
cggcagcctc ctagaaaagg cctggaatgg ctgggcgtga tctggggcag cgagacaacc 600
tactacaaca gcgccctgaa gtcccggctg accatcatca aggacaactc caagagccag 660
gtgttcctga agatgaacag cctgcagacc gacgacaccg ccatctacta ttgcgccaag 720
cactactact acggcggcag ctacgccatg gattattggg gccagggcac cagcgtgacc 780
gtttcttcta ccaccacacc agctcctaga cctccaactc ctgctcctac aatcgccagc 840
cagcctctgt ctctgaggcc agaagcttgt agacctgctg caggcggagc cgtgcataca 900
agaggactgg atttcgcctg cgacatctac atctgggccc ctctggctgg aacatgtggc 960
gtgctgctgc tgagcctggt catcaccctg tactgcagcc tgaagcgggg cagaaagaag 1020
ctgctgtaca tctttaagca gcccttcatg cggcccgtgc agaccacaca agaggaagat 1080
ggctgctcct gcagattccc cgaggaagaa gaaggcggct gcgagctgag agtgaagttc 1140
agccgttctg ccgacgctcc cgcctataag cagggacaga accagctgta caacgagctg 1200
aacctgggga gaagagaaga gtacgacgtg ctggacaagc ggagaggcag agatcctgag 1260
atgggcggca agcccagacg gaagaatcct caagagggcc tgtataatga gctgcagaaa 1320
gacaagatgg ccgaggccta cagcgagatc ggaatgaagg gcgagcgcag aagaggcaag 1380
ggacacgatg gactgtacca gggcctgagc accgccacca aggataccta tgatgccctg 1440
cacatgcagg ccctgcctcc aagatcaggc tctggttctg gcagcggcag catggtgtct 1500
aaaggcgagg aactgttcac cggcgtggtg cccattctgg tggaactgga cggggatgtg 1560
aacggccaca agtttagcgt tagcggcgaa ggcgaagggg atgccacata cggaaagctg 1620
accctgaagt tcatctgcac caccggcaag ctgcctgtgc cttggcctac actggtcacc 1680
acactgacat acggcgtgca gtgctttagc agataccccg accatatgaa gcagcacgac 1740
ttcttcaagt ccgccatgcc tgagggctac gtgcaagagc ggaccatctt ctttaaggac 1800
gacggcaact acaagaccag ggccgaagtg aagtttgagg gcgacaccct ggtcaaccgg 1860
atcgagctga agggcatcga cttcaaagag gatggcaaca tcctgggcca caagctcgag 1920
tacaactaca acagccacaa cgtgtacatc atggccgaca agcagaagaa cggcatcaag 1980
gccaacttca agatccggca caacatcgag gacggcagcg ttcagctggc cgatcactac 2040
cagcagaaca cccctatcgg agatggccct gtgctgctcc ccgacaatca ctacctgagc 2100
acacagagcg ccctgagcaa ggaccccaac gagaagaggg atcacatggt gctgctggaa 2160
tttgtgaccg ccgcaggcat caccctcggc atggacgaac tgtacaaa 2208
<210> 173
<211> 1115
<212> PRT
<213> 人工序列
<220>
<223> 合成肽
CD19scFv_CD8铰链TM_4-1BB_CD3ζ_EGFP-T2A-NKG2D.YA_CD8铰链TM
<400> 173
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 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
130 135 140
Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser
145 150 155 160
Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly
165 170 175
Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly
180 185 190
Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser
195 200 205
Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys
210 215 220
Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys
225 230 235 240
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly
245 250 255
Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro
260 265 270
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
275 280 285
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
290 295 300
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
305 310 315 320
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Ser Leu Lys Arg
325 330 335
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
340 345 350
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
355 360 365
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser Gly Ser Gly Ser Gly
485 490 495
Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile
500 505 510
Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser
515 520 525
Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe
530 535 540
Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
545 550 555 560
Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met
565 570 575
Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
580 585 590
Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
595 600 605
Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
610 615 620
Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
625 630 635 640
Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys
645 650 655
Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly
660 665 670
Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
675 680 685
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
690 695 700
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
705 710 715 720
Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
725 730 735
Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp
740 745 750
Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu
755 760 765
Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Leu Phe
770 775 780
Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys
785 790 795 800
Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp
805 810 815
Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn
820 825 830
Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys
835 840 845
Leu Val Lys Ser Ala His Trp Met Gly Leu Val His Ile Pro Thr Asn
850 855 860
Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu
865 870 875 880
Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser
885 890 895
Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys
900 905 910
Met Gln Arg Thr Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro
915 920 925
Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe
930 935 940
Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln
945 950 955 960
Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu
965 970 975
Lys Leu Val Lys Ser Ala His Trp Met Gly Leu Val His Ile Pro Thr
980 985 990
Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu
995 1000 1005
Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala
1010 1015 1020
Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr
1025 1030 1035
Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro Ala Pro Arg
1040 1045 1050
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
1055 1060 1065
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
1070 1075 1080
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
1085 1090 1095
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
1100 1105 1110
Tyr Cys
1115
<210> 174
<211> 3345
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码
CD19scFv_CD8铰链TM_4-1BB_CD3ζ_EGFP-T2A-NKG2D.YA_CD8铰链TM
<400> 174
atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60
cctgacatcc agatgaccca gacaaccagc agcctgtctg ccagcctggg cgatagagtg 120
accatcagct gtagagccag ccaggacatc agcaagtacc tgaactggta tcagcagaaa 180
cccgacggca ccgtgaagct gctgatctac cacaccagca gactgcacag cggcgtgcca 240
agcagatttt ctggcagcgg ctctggcacc gactacagcc tgacaatcag caacctggaa 300
caagaggata tcgctaccta cttctgccag caaggcaaca ccctgcctta cacctttggc 360
ggaggcacca agctggaaat cacaggcggc ggaggaagcg gaggcggagg atctggtggt 420
ggtggatctg aagtgaaact gcaagagtct ggccctggcc tggtggcccc atctcaatct 480
ctgagcgtga cctgtaccgt cagcggagtg tccctgcctg attatggcgt gtcctggatc 540
cggcagcctc ctagaaaagg cctggaatgg ctgggcgtga tctggggcag cgagacaacc 600
tactacaaca gcgccctgaa gtcccggctg accatcatca aggacaactc caagagccag 660
gtgttcctga agatgaacag cctgcagacc gacgacaccg ccatctacta ttgcgccaag 720
cactactact acggcggcag ctacgccatg gattattggg gccagggcac cagcgtgacc 780
gtttcttcta ccaccacacc agctcctaga cctccaactc ctgctcctac aatcgccagc 840
cagcctctgt ctctgaggcc agaagcttgt agacctgctg caggcggagc cgtgcataca 900
agaggactgg atttcgcctg cgacatctac atctgggccc ctctggctgg aacatgtggc 960
gtgctgctgc tgagcctggt catcaccctg tactgcagcc tgaagcgggg cagaaagaag 1020
ctgctgtaca tctttaagca gcccttcatg cggcccgtgc agaccacaca agaggaagat 1080
ggctgctcct gcagattccc cgaggaagaa gaaggcggct gcgagctgag agtgaagttc 1140
agccgttctg ccgacgctcc cgcctataag cagggacaga accagctgta caacgagctg 1200
aacctgggga gaagagaaga gtacgacgtg ctggacaagc ggagaggcag agatcctgag 1260
atgggcggca agcccagacg gaagaatcct caagagggcc tgtataatga gctgcagaaa 1320
gacaagatgg ccgaggccta cagcgagatc ggaatgaagg gcgagcgcag aagaggcaag 1380
ggacacgatg gactgtacca gggcctgagc accgccacca aggataccta tgatgccctg 1440
cacatgcagg ccctgcctcc aagatcaggc tctggttctg gcagcggcag catggtgtct 1500
aaaggcgagg aactgttcac cggcgtggtg cccattctgg tggaactgga cggggatgtg 1560
aacggccaca agtttagcgt tagcggcgaa ggcgaagggg atgccacata cggaaagctg 1620
accctgaagt tcatctgcac caccggcaag ctgcctgtgc cttggcctac actggtcacc 1680
acactgacat acggcgtgca gtgctttagc agataccccg accatatgaa gcagcacgac 1740
ttcttcaagt ccgccatgcc tgagggctac gtgcaagagc ggaccatctt ctttaaggac 1800
gacggcaact acaagaccag ggccgaagtg aagtttgagg gcgacaccct ggtcaaccgg 1860
atcgagctga agggcatcga cttcaaagag gatggcaaca tcctgggcca caagctcgag 1920
tacaactaca acagccacaa cgtgtacatc atggccgaca agcagaagaa cggcatcaag 1980
gccaacttca agatccggca caacatcgag gacggcagcg ttcagctggc cgatcactac 2040
cagcagaaca cccctatcgg agatggccct gtgctgctcc ccgacaatca ctacctgagc 2100
acacagagcg ccctgagcaa ggaccccaac gagaagaggg atcacatggt gctgctggaa 2160
tttgtgaccg ccgcaggcat caccctcggc atggacgaac tgtacaaact cgagggcggc 2220
ggagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctagg 2280
atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 2340
atcccactgt tcaaccagga ggttcagatt ccgctgaccg aaagctactg tggaccatgt 2400
cccaagaact ggatctgcta caaaaacaat tgttatcaat ttttcgacga aagcaagaat 2460
tggtacgaaa gccaggcttc atgcatgagc caaaatgcat ccctgctgaa ggtgtacagc 2520
aaggaggacc aggacctgct gaaacttgtc aaatccgctc actggatggg actggtccat 2580
attcctacaa atggatcctg gcagtgggag gatggctcta tcctgtctcc caatctcctg 2640
actatcatag agatgcagaa gggagattgt gcattgtatg ctagttcatt caagggatac 2700
attgaaaact gctcaacacc taatacctac atatgtatgc agcgcacagt gcagatccct 2760
ctgaccgaga gctactgtgg cccctgtcct aagaactgga tctgctacaa gaacaactgc 2820
taccagttct tcgacgagag caagaattgg tacgagagcc aggccagctg catgagccag 2880
aatgccagcc tgctgaaggt gtacagcaaa gaggaccagg atctgctgaa gctggtcaag 2940
agcgcccact ggatgggact cgtgcacatc cctacaaacg gcagctggca gtgggaggac 3000
ggctctatcc tgtctcctaa cctgctgacc atcatcgaga tgcagaaggg cgactgcgcc 3060
ctgtacgcca gcagctttaa gggctacatc gagaactgca gcacccctaa cacctacatc 3120
tgtatgcagc ggaccgtgac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 3180
atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 3240
gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 3300
acttgtgggg tccttctcct gtcactggtt atcacccttt actgc 3345
<210> 175
<211> 161
<212> PRT
<213> 人工序列
<220>
<223> 合成肽FLAG_6xHis_mutIL2
<400> 175
Gly Ser Ser Gly Ser Ser Asp Tyr Lys Asp Asp Asp Asp Lys His His
1 5 10 15
His His His His His His Gly Ser Ser Gly Ser Ser Ala Pro Thr Ser
20 25 30
Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp
35 40 45
Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu
50 55 60
Thr Ala Met Leu Thr Lys Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu
65 70 75 80
Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu
85 90 95
Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp
100 105 110
Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu
115 120 125
Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu
130 135 140
Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu
145 150 155 160
Thr
<210> 176
<211> 483
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码FLAG_6xHis_mutIL2
<400> 176
ggaagtagcg gtagtagtga ttacaaggac gatgacgaca agcaccacca tcatcatcat 60
caccacggta gcagcggcag cagtgccccc acctctagca gcacaaagaa gacccagctg 120
caactggaac acctcctgct ggacctgcag atgatcctga acggcatcaa caactacaag 180
aaccccaagc tgaccgccat gctgaccaaa aagttttaca tgcccaagaa ggccaccgag 240
cttaaacacc tgcaatgcct tgaggaggag ctgaagcccc tggaggaggt actgaacctg 300
gcccagagca agaactttca tctgaggccc agggacctga ttagcaacat caacgtgatc 360
gtgttggagt tgaagggcag cgagaccacg ttcatgtgcg agtacgccga cgagacggcc 420
accatagtgg agtttcttaa caggtggatc accttctcac agtctatcat cagcaccctg 480
acc 483
<210> 177
<211> 343
<212> PRT
<213> 人工序列
<220>
<223> 合成肽ULBP2.R80W_FLAG_6xHis_mutIL2
<400> 177
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 Gly Thr Gly Ser Ser Gly Ser Ser Asp Tyr Lys Asp
180 185 190
Asp Asp Asp Lys His His His His His His His His Gly Ser Ser Gly
195 200 205
Ser Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu
210 215 220
Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn
225 230 235 240
Tyr Lys Asn Pro Lys Leu Thr Ala Met Leu Thr Lys Lys Phe Tyr Met
245 250 255
Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu
260 265 270
Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe
275 280 285
His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu
290 295 300
Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu
305 310 315 320
Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln
325 330 335
Ser Ile Ile Ser Thr Leu Thr
340
<210> 178
<211> 1029
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码ULBP2.R80W_FLAG_6xHis_mutIL2
<400> 178
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
ggtaccggta gtagcggtag tagtgattac aaggacgatg acgacaagca ccaccatcat 600
catcatcacc acggtagcag cggcagcagt gcccccacct ctagcagcac aaagaagacc 660
cagctgcaac tggaacacct cctgctggac ctgcagatga tcctgaacgg catcaacaac 720
tacaagaacc ccaagctgac cgccatgctg accaaaaagt tttacatgcc caagaaggcc 780
accgagctta aacacctgca atgccttgag gaggagctga agcccctgga ggaggtactg 840
aacctggccc agagcaagaa ctttcatctg aggcccaggg acctgattag caacatcaac 900
gtgatcgtgt tggagttgaa gggcagcgag accacgttca tgtgcgagta cgccgacgag 960
acggccacca tagtggagtt tcttaacagg tggatcacct tctcacagtc tatcatcagc 1020
accctgacc 1029
<210> 179
<211> 343
<212> PRT
<213> 人工序列
<220>
<223> 合成肽ULBP2.S2_FLAG_6xHis_mutIL2
<400> 179
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 Arg Ile 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 Gly Thr Gly Ser Ser Gly Ser Ser Asp Tyr Lys Asp
180 185 190
Asp Asp Asp Lys His His His His His His His His Gly Ser Ser Gly
195 200 205
Ser Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu
210 215 220
Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn
225 230 235 240
Tyr Lys Asn Pro Lys Leu Thr Ala Met Leu Thr Lys Lys Phe Tyr Met
245 250 255
Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu
260 265 270
Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe
275 280 285
His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu
290 295 300
Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu
305 310 315 320
Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln
325 330 335
Ser Ile Ile Ser Thr Leu Thr
340
<210> 180
<211> 1029
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码ULBP2.S2_FLAG_6xHis_mutIL2
<400> 180
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 gcgactctga tgaggatttg gtcgatggga 480
gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540
ggtaccggta gtagcggtag tagtgattac aaggacgatg acgacaagca ccaccatcat 600
catcatcacc acggtagcag cggcagcagt gcccccacct ctagcagcac aaagaagacc 660
cagctgcaac tggaacacct cctgctggac ctgcagatga tcctgaacgg catcaacaac 720
tacaagaacc ccaagctgac cgccatgctg accaaaaagt tttacatgcc caagaaggcc 780
accgagctta aacacctgca atgccttgag gaggagctga agcccctgga ggaggtactg 840
aacctggccc agagcaagaa ctttcatctg aggcccaggg acctgattag caacatcaac 900
gtgatcgtgt tggagttgaa gggcagcgag accacgttca tgtgcgagta cgccgacgag 960
acggccacca tagtggagtt tcttaacagg tggatcacct tctcacagtc tatcatcagc 1020
accctgacc 1029
<210> 181
<211> 412
<212> PRT
<213> 人工序列
<220>
<223> 合成肽ULBP2.R80W_Fc1
<400> 181
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 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
180 185 190
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
195 200 205
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
210 215 220
Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe
225 230 235 240
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
245 250 255
Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
260 265 270
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
275 280 285
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
290 295 300
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
305 310 315 320
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
325 330 335
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
340 345 350
Glu Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
355 360 365
Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
370 375 380
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
385 390 395 400
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
405 410
<210> 182
<211> 1236
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码ULBP2.R80W_Fc1
<400> 182
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
gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 600
ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 660
acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 720
aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 780
tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 840
gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 900
atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 960
gacgagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 1020
gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactatga cactaccccg 1080
cccgtgctgg attccgacgg aagcttcttc ctgtactccg acctgaccgt ggacaagtcg 1140
agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 1200
tacacccaga agtcactgag cctctccccc ggaaag 1236
<210> 183
<211> 368
<212> PRT
<213> 人工序列
<220>
<223> 合成肽Fc2_mutIL2
<400> 183
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser
225 230 235 240
Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu
245 250 255
Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr
260 265 270
Ala Met Leu Thr Lys Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu
275 280 285
Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val
290 295 300
Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu
305 310 315 320
Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr
325 330 335
Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe
340 345 350
Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr
355 360 365
<210> 184
<211> 1104
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码Fc2_mutIL2
<400> 184
gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60
ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120
acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180
aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240
tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300
gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360
atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420
aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480
gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540
cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600
agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660
tacacccaga agtcactgag cctctccccc ggaggaggtg gcagcgcccc cacctctagc 720
agcacaaaga agacccagct gcaactggaa cacctcctgc tggacctgca gatgatcctg 780
aacggcatca acaactacaa gaaccccaag ctgaccgcca tgctgaccaa aaagttttac 840
atgcccaaga aggccaccga gcttaaacac ctgcaatgcc ttgaggagga gctgaagccc 900
ctggaggagg tactgaacct ggcccagagc aagaactttc atctgaggcc cagggacctg 960
attagcaaca tcaacgtgat cgtgttggag ttgaagggca gcgagaccac gttcatgtgc 1020
gagtacgccg acgagacggc caccatagtg gagtttctta acaggtggat caccttctca 1080
cagtctatca tcagcaccct gacc 1104
<210> 185
<211> 349
<212> PRT
<213> 人工序列
<220>
<223> 合成肽Fc2_mutIL15
<400> 185
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Asn Trp Val Asn Val
225 230 235 240
Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile
245 250 255
Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val
260 265 270
Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu
275 280 285
Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu
290 295 300
Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys
305 310 315 320
Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln
325 330 335
Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser
340 345
<210> 186
<211> 1047
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码Fc2_mutIL15
<400> 186
gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60
ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120
acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180
aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240
tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300
gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360
atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420
aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480
gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540
cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600
agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660
tacacccaga agtcactgag cctctccccc ggaggaggtg gcagcaattg ggtgaacgtg 720
atttctgact tgaagaaaat cgaggacttg attcaaagta tgcacataga cgcaacgctc 780
tatactgaga gtgatgttca tccctcttgt aaagttaccg ctatgaagtg tttcttgctc 840
gaactccaag ttatcagtct ggagagcggt gatgcctcca tacacgatac agtcgaaaac 900
cttatcattc tcgcaaataa ctcattgagt agcaatggca atgttacaga gtctgggtgt 960
aaggaatgcg aagaacttga agagaaaaac ataaaggagt tcctccaatc attcgtgcat 1020
attgtccaga tgtttatcaa cactagc 1047
<210> 187
<211> 412
<212> PRT
<213> 人工序列
<220>
<223> 合成肽ULBP2.S2_Fc1
<400> 187
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 Arg Ile 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 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
180 185 190
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
195 200 205
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
210 215 220
Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe
225 230 235 240
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
245 250 255
Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
260 265 270
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
275 280 285
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
290 295 300
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
305 310 315 320
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
325 330 335
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
340 345 350
Glu Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
355 360 365
Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
370 375 380
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
385 390 395 400
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
405 410
<210> 188
<211> 1236
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码ULBP2.S2_Fc1
<400> 188
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 gcgactctga tgaggatttg gtcgatggga 480
gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540
gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 600
ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 660
acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 720
aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 780
tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 840
gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 900
atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 960
gacgagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 1020
gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactatga cactaccccg 1080
cccgtgctgg attccgacgg aagcttcttc ctgtactccg acctgaccgt ggacaagtcg 1140
agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 1200
tacacccaga agtcactgag cctctccccc ggaaag 1236
<210> 189
<211> 412
<212> PRT
<213> 人工序列
<220>
<223> 合成肽ULBP2.S3_Fc1
<400> 189
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 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
180 185 190
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
195 200 205
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
210 215 220
Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe
225 230 235 240
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
245 250 255
Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
260 265 270
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
275 280 285
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
290 295 300
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
305 310 315 320
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
325 330 335
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
340 345 350
Glu Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
355 360 365
Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
370 375 380
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
385 390 395 400
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
405 410
<210> 190
<211> 1236
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码ULBP2.S3_Fc1
<400> 190
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
gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 600
ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 660
acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 720
aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 780
tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 840
gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 900
atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 960
gacgagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 1020
gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactatga cactaccccg 1080
cccgtgctgg attccgacgg aagcttcttc ctgtactccg acctgaccgt ggacaagtcg 1140
agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 1200
tacacccaga agtcactgag cctctccccc ggaaag 1236
<210> 191
<211> 368
<212> PRT
<213> 人工序列
<220>
<223> 合成肽Fc2_wtIL2 (野生型)
<400> 191
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser
225 230 235 240
Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu
245 250 255
Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr
260 265 270
Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu
275 280 285
Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val
290 295 300
Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu
305 310 315 320
Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr
325 330 335
Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe
340 345 350
Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile Ile Ser Thr Leu Thr
355 360 365
<210> 192
<211> 1104
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码Fc2_wtIL2
<400> 192
gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60
ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120
acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180
aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240
tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300
gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360
atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420
aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480
gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540
cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600
agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660
tacacccaga agtcactgag cctctccccc ggaggaggtg gcagcgctcc aacctctagc 720
tctacgaaga aaactcaact ccagcttgag catttgcttt tggaccttca aatgatattg 780
aacgggatta acaattacaa gaaccccaaa ctgacgcgaa tgcttacatt caaattttac 840
atgccaaaga aggccaccga gctgaaacac ttgcaatgtc ttgaggaaga gctgaagcca 900
ttggaggagg tcctgaattt ggctcaaagt aagaacttcc acctgcggcc aagggatctc 960
ataagcaaca taaatgtcat tgtattggaa ctgaaaggga gcgaaaccac ctttatgtgc 1020
gagtatgcag atgaaactgc aaccatagtc gagttcctca atcgctggat aactttcgcg 1080
cagtcaatta tatcaacact cacc 1104
<210> 193
<211> 368
<212> PRT
<213> 人工序列
<220>
<223> 合成肽Fc2_IL21.wt (野生型)
<400> 193
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gln Gly Gln Asp Arg
225 230 235 240
His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys
245 250 255
Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp
260 265 270
Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala
275 280 285
Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val
290 295 300
Leu Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg
305 310 315 320
Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys
325 330 335
Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys
340 345 350
Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser
355 360 365
<210> 194
<211> 1104
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码Fc2_IL21.wt (野生型)
<400> 194
gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60
ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120
acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180
aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240
tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300
gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360
atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420
aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480
gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540
cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600
agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660
tacacccaga agtcactgag cctctccccc ggaggaggtg gcagccaggg tcaagaccgc 720
catatgatcc gaatgcgaca gctcattgat attgtcgatc aattgaaaaa ttacgtgaat 780
gatcttgtac cggagttcct ccccgcaccg gaggacgttg aaacgaattg tgagtggtca 840
gcattttctt gctttcagaa ggctcaactc aagagtgcaa acacgggtaa caacgagcgc 900
attatcaatg ttctcatcaa aaagctgaaa cgaaaaccgc ctagcaccaa cgcaggcaga 960
cgacagaagc accggctcac gtgcccaagt tgcgattctt atgagaaaaa gccaccgaaa 1020
gaattcctgg agcggttcaa gtccctcttg cagaaaatga ttcatcagca tctctccagc 1080
aggacacacg gctccgagga ctcc 1104
<210> 195
<211> 368
<212> PRT
<213> 人工序列
<220>
<223> 合成肽Fc2_IL21.D18A
<400> 195
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gln Gly Gln Asp Arg
225 230 235 240
His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Ala Gln Leu Lys
245 250 255
Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp
260 265 270
Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala
275 280 285
Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val
290 295 300
Leu Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg
305 310 315 320
Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys
325 330 335
Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys
340 345 350
Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser
355 360 365
<210> 196
<211> 1104
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码Fc2_IL21.D18A
<400> 196
gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60
ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120
acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180
aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240
tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300
gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360
atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420
aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480
gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540
cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600
agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660
tacacccaga agtcactgag cctctccccc ggaggaggtg gcagccaggg tcaagaccgc 720
catatgatcc gaatgcgaca gctcattgat attgtcgcac aattgaaaaa ttacgtgaat 780
gatcttgtac cggagttcct ccccgcaccg gaggacgttg aaacgaattg tgagtggtca 840
gcattttctt gctttcagaa ggctcaactc aagagtgcaa acacgggtaa caacgagcgc 900
attatcaatg ttctcatcaa aaagctgaaa cgaaaaccgc ctagcaccaa cgcaggcaga 960
cgacagaagc accggctcac gtgcccaagt tgcgattctt atgagaaaaa gccaccgaaa 1020
gaattcctgg agcggttcaa gtccctcttg cagaaaatga ttcatcagca tctctccagc 1080
aggacacacg gctccgagga ctcc 1104
<210> 197
<211> 368
<212> PRT
<213> 人工序列
<220>
<223> 合成肽Fc2_IL21.E109R
<400> 197
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gln Gly Gln Asp Arg
225 230 235 240
His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys
245 250 255
Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp
260 265 270
Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala
275 280 285
Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val
290 295 300
Leu Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg
305 310 315 320
Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys
325 330 335
Lys Pro Pro Lys Glu Phe Leu Arg Arg Phe Lys Ser Leu Leu Gln Lys
340 345 350
Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser
355 360 365
<210> 198
<211> 1104
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码Fc2_IL21.E109R
<400> 198
gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60
ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120
acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180
aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240
tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300
gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360
atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420
aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480
gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540
cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600
agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660
tacacccaga agtcactgag cctctccccc ggaggaggtg gcagccaggg tcaagaccgc 720
catatgatcc gaatgcgaca gctcattgat attgtcgatc aattgaaaaa ttacgtgaat 780
gatcttgtac cggagttcct ccccgcaccg gaggacgttg aaacgaattg tgagtggtca 840
gcattttctt gctttcagaa ggctcaactc aagagtgcaa acacgggtaa caacgagcgc 900
attatcaatg ttctcatcaa aaagctgaaa cgaaaaccgc ctagcaccaa cgcaggcaga 960
cgacagaagc accggctcac gtgcccaagt tgcgattctt atgagaaaaa gccaccgaaa 1020
gaattcctgc ggcggttcaa gtccctcttg cagaaaatga ttcatcagca tctctccagc 1080
aggacacacg gctccgagga ctcc 1104
<210> 199
<211> 368
<212> PRT
<213> 人工序列
<220>
<223> 合成肽Fc2_IL21.D18A.E109R
<400> 199
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gln Gly Gln Asp Arg
225 230 235 240
His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Ala Gln Leu Lys
245 250 255
Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp
260 265 270
Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala
275 280 285
Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val
290 295 300
Leu Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg
305 310 315 320
Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys
325 330 335
Lys Pro Pro Lys Glu Phe Leu Arg Arg Phe Lys Ser Leu Leu Gln Lys
340 345 350
Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser
355 360 365
<210> 200
<211> 1104
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码Fc2_IL21.D18A.E109R
<400> 200
gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60
ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120
acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180
aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240
tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300
gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360
atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420
gacgagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480
gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540
cccgtgctgg attccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600
agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660
tacacccaga agtcactgag cctctccccc ggaggaggtg gcagccaggg tcaagaccgc 720
catatgatcc gaatgcgaca gctcattgat attgtcgcac aattgaaaaa ttacgtgaat 780
gatcttgtac cggagttcct ccccgcaccg gaggacgttg aaacgaattg tgagtggtca 840
gcattttctt gctttcagaa ggctcaactc aagagtgcaa acacgggtaa caacgagcgc 900
attatcaatg ttctcatcaa aaagctgaaa cgaaaaccgc ctagcaccaa cgcaggcaga 960
cgacagaagc accggctcac gtgcccaagt tgcgattctt atgagaaaaa gccaccgaaa 1020
gaattcctgc ggcggttcaa gtccctcttg cagaaaatga ttcatcagca tctctccagc 1080
aggacacacg gctccgagga ctcc 1104
<210> 201
<211> 233
<212> PRT
<213> 人工序列
<220>
<223> 合成肽Fc2
<400> 201
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 Ala 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 Ala 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 Lys 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 Lys 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
225 230
<210> 202
<211> 699
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸，编码Fc2
<400> 202
atggacccca aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60
ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120
cgcacccctg aagtgacctg tgtcgtcgtg gcagtgtcac acgaggaccc ggaggtcaag 180
ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240
cagtacgcct cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300
aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360
accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420
cggaaggagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480
tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540
ccgcccgtgc tgaagtccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600
tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660
cactacaccc agaagtcact gagcctctcc cccggaaag 699

Claims (11)

1.一种非天然的、经修饰的NKG2D受体，其中所述受体结合NKG2D配体的非天然的、经修饰的α1-α2结构域，但不结合NKG2D的天然配体，其中附着于哺乳动物细胞的所述经修饰的NKG2D受体用作替代高亲和力受体，用于向所述细胞递送附着于所述经修饰的α1-α2结构域的异源原子或异源分子，并且其中所述经修饰的NKG2D受体不直接激活所述哺乳动物细胞或不直接向所述哺乳动物细胞传导信号。
2.根据权利要求1所述的异源分子，其中所述异源分子当通过所述经修饰的α1-α2结构域递送至所述哺乳动物细胞时，能够通过所述哺乳动物细胞上或所述哺乳动物细胞内的其各自的受体亚基激活所述细胞或向所述细胞传导信号。
3.根据权利要求1所述的异源原子或异源分子，其中所述异源原子或异源分子当通过所述经修饰的α1-α2结构域递送至所述哺乳动物细胞时，能够直接影响所述哺乳动物细胞，而不依赖于除所述非天然的、经修饰的NKG2D受体之外的所述细胞上或所述细胞内的任何受体。
4.根据权利要求1所述的异源原子或异源分子，其中所述异源原子或异源分子当通过所述经修饰的α1-α2结构域递送至所述哺乳动物细胞时，能够被所述哺乳动物细胞内化，其中与所述经修饰的α1-α2结构域结合或未结合的被内化的异源原子或异源分子影响所述哺乳动物细胞。
5.根据权利要求1所述的异源原子或异源分子，其中所述异源原子或异源分子当通过所述经修饰的α1-α2结构域递送至所述哺乳动物细胞时，能够给所述哺乳动物细胞提供通过体内、离体或体外检测或者通过成像系统可检测的标志物。
6.根据权利要求1所述的非天然的、经修饰的NKG2D受体，其在细胞内附着有共刺激，其中所述共刺激结构域增强被引入所述哺乳动物细胞的效应分子的功能，其中所述效应分子不与经修饰的α1-α2结构域附着。
7.根据权利要求3所述的异源原子或异源分子，其中所述异源原子或异源分子当通过所述经修饰的α1-α2结构域递送至所述哺乳动物细胞时，能够通过发射能量或破坏细胞膜，例如促进形成补体依赖性大分子攻击复合物，来损伤所述细胞。
8.一种附着于哺乳动物细胞的非天然的、经修饰的NKG2D受体，其中在不存在活性CD3-ζ结构域的情况下，共刺激结构域在细胞内与所述经修饰的NKG2D受体附着，其中所述受体结合NKG2D配体的非天然的、经修饰的α1-α2结构域，但不结合NKG2D的天然配体。
9.根据权利要求8所述的非天然的、经修饰的α1-α2结构域，其上附着有异源原子或异源分子，其中所述异源原子或异源分子当通过经修饰的α1-α2结构域递送至所述哺乳动物细胞时，能够被所述哺乳动物细胞内化，其中与所述经修饰的α1-α2结构域结合或未结合的被内化的异源原子或异源分子影响所述哺乳动物细胞。
10.根据权利要求8所述的非天然的、经修饰的α1-α2结构域，其上附着有异源原子或异源分子，其中所述异源原子或异源分子向所述哺乳动物细胞提供靶向或归巢功能，所述靶向或归巢功能促进所述哺乳动物细胞向携带靶标的细胞或携带靶标的表面的递送，当所述哺乳动物细胞到达其靶向表面或环境时不直接导致所述哺乳动物细胞的活化。
11.根据权利要求8所述的NKG2D-附着的共刺激结构域，其中所述共刺激结构域增强被引入所述哺乳动物细胞的效应分子的功能，其中所述效应分子不与经修饰的α1-α2结构域附着。
CN201980087716.0A 2018-11-05 2019-11-05 不直接向其附着的细胞传导信号的非天然nkg2d受体 Pending CN113614103A (zh)

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EP3877403A4 (en) 2022-08-17
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CO2021007302A2 (es) 2021-08-30
CA3118498A1 (en) 2020-05-14
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