CN113493518B - 优化的连接肽组合及其应用 - Google Patents

优化的连接肽组合及其应用 Download PDF

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CN113493518B
CN113493518B CN202010254451.3A CN202010254451A CN113493518B CN 113493518 B CN113493518 B CN 113493518B CN 202010254451 A CN202010254451 A CN 202010254451A CN 113493518 B CN113493518 B CN 113493518B
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黄霞
赵永春
陈雪娇
赵文旭
陈军
徐艳敏
齐亚男
单娟娟
张巍
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Chongqing Precision Biotech Co ltd
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Abstract

本发明属于基因工程技术领域，本发明属于基因工程技术领域，具体涉及一种优化的连接肽和其连接的双特异性单链抗体，以及包含双特异性单链抗体的CAR结构、表达载体、免疫细胞和应用。所述双特异性单链抗体中，由两种linker进行连接两种单链抗体的重链或轻链，所述CAR结构的构建克服现有技术中双靶点CAR的技术效果缺陷，可同时靶向两个不同的肿瘤抗原，提高对肿瘤细胞杀伤，减少其免疫逃逸的几率。

Description

优化的连接肽组合及其应用
技术领域
本发明属于基因工程技术领域，具体涉及一种优化的连接肽和其连接的双特异性单链抗体，以及包含双特异性单链抗体的CAR结构、表达载体、免疫细胞和应用。
背景技术
CAR-T(嵌合抗原受体T)细胞疗法在治疗血液系统恶性肿瘤中已取得了显著成果。但是由于肿瘤尤其是实体瘤的异质性，往往肿瘤细胞表面不止表达一个靶抗原，并且随着研究的进展，研发人员还发现经CAR-T细胞治疗的少部分患者出现肿瘤细胞下调或突变以及其表面表达的CAR-T靶点抗原产生逃逸(Robbie G.Majzner et al.(2018)TumorAntigen Escape from CAR T-cell Therapy)，最终造成治疗效果较差以及复发等情况。因此设计针对多靶点的CAR结构十分有必要，若简单的同时表达2个不同靶点的CAR，但不同的CAR结构感染能力不同，会产生多种CAR-T细胞亚群，不利于产品临床应用；如果仅是简单的两个不同靶点CAR结构串联于一个载体，这样CAR载体较大不易转导T细胞，且不同的结构涉及到CAR载体病毒制备能力不同，CAR转导能力不同，对T细胞产生的刺激不同，疗效不同等诸多因素。因此，为了克服现有技术中双靶点CAR的技术效果缺陷，本发明提出了新的解决方案。
发明内容
有鉴于此，本发明目的在于提供一种双特异性单链抗体。
所述双特异抗体包含识别两个不同抗原或同一抗原两个不同表位的ScFv重链和轻链，所述重链/轻链和轻链/重链通过Linker1或Linker2连接，linker1结构的氨基酸序列如SEQ ID NO:17或其功能性变体所示，Linker2结构的氨基酸序列如SEQ ID NO:18或其功能性变体所示。
具体地，所述linker1连接不同抗原的轻链或重链，所述linker2连接同一抗原的轻链或重链。
进一步，编码所述linker1的核苷酸序列如SEQ ID NO:1所示，编码所述Linker2结构的核苷酸序列如SEQ ID NO:2所示，或其功能性变体。
进一步，所述双特异性单链抗体结构为VL(ScFv1)-linker1-VL(ScFv2)-linker2-VH(ScFv2)-linker1-VH(ScFv1)或VL(ScFv1)-linker1-VH(ScFv2)-linker2-VL(ScFv2)-linker1-VH(ScFv1)或VH(ScFv1)-linker1-VL(ScFv2)-linker2-VH(ScFv2)-linker1-VL(ScFv1)。
进一步，所述ScFv1来源任选于抗体CD19、CD123、MOv-γ、PSMA、IL13Rα2、EGFRvIII、EGFR、EPCAM、GD2、MUC1、HER2、GPC3、CEA、Meso、CD133、NKG2D、CD138、LeY、k-Light、CD33、ROR1、BCMA、CD30、CD20、CD22、PSCA、CLL-1、CD70中一种；所述ScFv2来源任选于抗体CD19、CD123、MOv-γ、PSMA、IL13Rα2、EGFRvIII、EGFR、EPCAM、GD2、MUC1、HER2、GPC3、CEA、Meso、CD133、NKG2D、CD138、LeY、k-Light、CD33、ROR1、BCMA、CD30、CD20、CD22、PSCA、CLL-1、CD70中一种。
优选地，所述双特异性单链抗体结构为抗CD19ScFv轻链可变区-Linker1-抗CD123ScFv轻链可变区-Linker2-抗CD123ScFv重链可变区-Linker1-抗CD19ScFv重链可变区或抗CD123ScFv轻链可变区-Linker1-抗CD19ScFv轻链可变区-Linker2-抗CD19ScFv重链可变区-Linker1-抗CD123ScFv重链可变区或CD19的ScFv的轻链可变区-Linker1-抗CD22的重链可变区-Linker2-抗CD22的轻链可变区-Linker1-抗CD19的ScFv的重链可变区或CD22的ScFv的重链可变区-Linker1-抗CD19的轻链可变区-Linker2-抗CD19的重链可变区-Linker1-抗CD22的ScFv的轻链可变区。
进一步，所述抗CD19ScFv轻链可变区核苷酸序列如SEQ ID NO:8所示，氨基酸序列如SEQ ID NO:19所示；抗CD19ScFv重链可变区核苷酸序列如SEQ ID NO:7所示，氨基酸序列如SEQ ID NO:20所示；抗CD123ScFv重链可变区核苷酸序列如SEQ ID NO:11所示，氨基酸序列如SEQ ID NO:24所示；抗CD123ScFv轻链可变区核苷酸序列如SEQ ID NO:12所示，氨基酸序列如SEQ ID NO:23所示；抗CD22的重链可变区核苷酸序列如SEQ ID NO:9所示，氨基酸序列如SEQ ID NO:21所示；抗CD22的轻链可变区核苷酸序列如SEQ ID NO:10所示，氨基酸序列如SEQ ID NO:22所示；
本发明目的在于还提供了一种包含前述双特异性单链抗体的CAR。
进一步，所述CAR还包含铰链结构、跨膜结构、胞内共刺激结构域和胞内活化信号。
进一步，所述CAR中的铰链区序列可以来源于：IgG、CD8、CD7、CD4；CAR结构中跨膜区可以来源于：CD8、CD28、CD3ε、CD4、CD16、CD137、CD80以及CD86；CAR结构中胞内信号可来源于：CD3、CD137、CD28、CD27、OX40、ICOS、GITR、CD2、CD40、PD-1、PD1L、B7-H3、淋巴细胞功能相关抗原-1(LFA-1)、ICAM-1、CD7、NKG2C、CD83、CD86以及CD127。
进一步，所述胞内信号包含胞内共刺激结构域和胞内活化信号。
优选地，所述CAR结构包含：CD8铰链区，CD8跨膜区、BBZ来源CD137的胞内共刺激信号和CD3ζ胞内活化信号，所述CD8铰链区核苷酸序列如SEQ ID NO:13所示，氨基酸序列如SEQ ID NO:29所示；所述CD8跨膜区的核苷酸序列如SEQ ID NO:14所示，氨基酸序列如SEQID NO:30；所述CD137胞内共刺激信号核苷酸序列如SEQ ID NO:15所示，氨基酸序列如SEQID NO:31所示；所述CD3ζ胞内活化信号核苷酸序列如SEQ ID NO:16所示，氨基酸序列如SEQID NO:32所示。
进一步，所述的CAR的氨基酸序列如SEQ ID NO:25或SEQ ID NO:26或SEQ ID NO:27或SEQ ID NO:28所示，或其功能性变体。
进一步，所述的CAR的核苷酸序列如SEQ ID NO:3或SEQ ID NO:4或SEQ ID NO:5或SEQ ID NO:6所示，或其功能性变体。
在某些实施例中，抗原识别区可以为识别靶抗原的配体/受体；在某些实施例中，ScFv可以是鼠源抗体或全人源抗体或人鼠嵌合抗体的ScFv，也可以是鲨鱼、羊驼或骆驼抗体等单域抗体，也可以是人工设计的识别特定靶点的靶点特异性纤连蛋白III型(FN3)结构域组合。
具体的，所述“功能性变体”通常是指包括与其具有基本上相同的功能(例如，可以具备所述嵌合抗原受体的性质)，且与其具有至少85％(例如，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的氨基酸序列。在某些实施方式中，所述氨基酸序列的变体为与其具有基本上相同的功能。
本发明目的在于还提供了一种包含前述CAR的表达载体。
进一步，所述表达载体慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体、质粒中的任一种。
在某些实施例中，所述慢病毒载体选自基本上由以下组成的群组：人免疫缺陷病毒1(HIV-1)、人免疫缺陷病毒2(HIV-2)、维斯纳-梅迪病毒(vi sna-maedi virus，VMV)病毒、山羊关节炎-脑炎病毒(CAEV)、马传染性贫血病毒(EIAV)、猫免疫缺陷病毒(FIV)、牛免疫缺陷病毒(BIV)和猿猴免疫缺陷病毒(SIV)。
在某些实施例中，载体包含左(5')逆转录病毒LTR、Psi(Ψ)包装信号、中心多嘌呤段/DNA瓣(cPPT/FLAP)、逆转录病毒导出元件、可操作地连接到编码本文所涵盖的CAR的多核苷酸的启动子和右(3')逆转录病毒LTR。
在某些实施例中，CAR包含乙型肝炎病毒转录后调节元件(HPRE)或土拔鼠转录后调节元件(WPRE)以及优化的土拔鼠转录后调节元件(oPRE)。
本发明目的在于还提供了一种包含前述表达载体的免疫细胞。
优选地，所述免疫细胞为T细胞、B细胞、NK细胞、NKT细胞、DC细胞、巨噬细胞中一种。
更优选地，所述免疫细胞为T细胞。
具体的，所述细胞可以与其他可增强CAR表达活性的活性剂和/或治疗组合使用。
具体的，所述活性剂和/或治疗可以是手术、化疗、放射、免疫抑制剂，例如环孢素(cyclosporin)、硫唑嘌呤(azathioprine)、甲氨蝶呤(methotrexate)、霉酚酸酯(mycophenolate)和FK506、抗体或其它免疫清除剂(immunoablativeagents)例如CAMPATH、抗CD3抗体或其它抗体治疗、环磷酰胺(cytoxan)、氟达拉滨(fludarabine)、环孢素(cyclosporin)、FK506、雷帕霉素(rapamycin)、霉酚酸(mycophenolicacid)、类固醇(steroids)、FR901228、细胞因子和辐射。
在某些实施例中，所述细胞可以表达其它活性剂，例如，增强CAR表达细胞活性的活性剂。活性剂可以是阻断抑制性分子的活性剂。抑制性分子如PD1可以在一些实施方案中降低CAR表达细胞发动免疫效应子反应的能力。抑制性分子包括PD1、PD-L1、CTLA4、TIM3、LAG3、VISTA、BTLA、TIGIT,LAIR1、CD160、2B4、CEACAM(CEACAM-1、CEACAM-3、CEACAM-5)、LAG3、VISTA、BTLA、TIG、LAIR1、CD160、2B4、CD80、CD86、B7-H3(CD276)、B7-H4(VTCN1)、HVEM(TNFRSF14或CD270)、KIR、A2aR、MHC I类、MHC II类、GAL9、腺苷、TGFR(TGFRβ)和TGFRβ。所述抑制性分子的胞外结构域可以融合到跨膜结构域和胞内信号传导结构域，比如PD1 CAR。
本发明目的在于还提供了提升识别前述的靶点效应细胞的方法，所述方法为前述的表达载体转导免疫细胞。
进一步，所述免疫细胞为T细胞、B细胞、NK细胞、NKT细胞、DC细胞、巨噬细胞中一种。
优选地，所述免疫细胞为T细胞。
本发明目的在于还提供了一中前述的linker1或前述的双特异性单链抗体或前述的CAR或前述的表达载体在制备肿瘤药物中的应用。
进一步，所述肿瘤为恶性肿瘤，包括急性淋巴样白血病、慢性淋巴细胞白血病、慢性髓性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、前列腺癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、胰腺癌、肺癌、肾癌、肝癌、脑癌和皮肤癌；所述肿瘤高表达CD19、CD20、CD123、CD22、BCMA、ROR1、CEA、间皮素、PSCA、PSMA、c-Met、GPC-3、Her2、EGFRvIII、GD-2、NY-ESO-1TCR、MAGE A3TCR中的一种或多种。
本发明的有益效果：
本发明为克服现有技术中双靶点CAR的技术效果缺陷，构建新的CAR结构的构建方案，包含所述构建的CAR结构的CAR-T细胞可同时靶向两个不同的肿瘤抗原，提高对肿瘤细胞杀伤，减少其免疫逃逸的几率，降低CAR-T治疗后肿瘤复发率，且CAR转导效率高，适合工业化生产；经试验验证可同时靶向CD19和CD22的双特异性位点和同时靶向CD19和CD123的CAR-T细胞均具有很好的抗肿瘤效果。
附图说明
图1a为一种双CAR结构图。
图1b为另一种双CAR结构图。
图1a1为CD19和CD123双CAR结构图。
图1b1为CD19和CD22双CAR结构图。
图2a为CD19和CD123双靶点CAR结构在CHO细胞表达检测。
图2b为CD19和CD22双靶点CAR结构在CHO细胞表达检测。
图3a为CD19和CD123靶细胞抗原表达。
图3b为CD19和CD22靶细胞抗原表达。
图4a为CD19和CD123双靶点CAR结构在T淋巴细胞中表达检测。
图4b为CD19和CD22双靶点CAR结构在T淋巴细胞中表达检测。
图5a为CD19和CD123双靶点CAR体外杀伤结果。
图5b为CD19和CD22双靶点CAR体外杀伤结果。
图6a为小鼠荷瘤CD19和CD123靶细胞表型检测。
图6b为小鼠荷瘤CD19和CD22靶细胞表型检测。
图7a为CD19和CD123双靶点CAR体内杀伤结果。
图7b为CD19和CD22双靶点CAR体内杀伤结果。
图8a为CD19和CD123双靶点CAR回输后荷瘤小鼠生存曲线。
图8b为CD19和CD22双靶点CAR回输后荷瘤小鼠生存曲线。
具体实施方式
以下将参照附图，对本发明的优选实施例进行详细描述。优选实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南(第三版，J.萨姆布鲁克等著)中所述的条件，或按照制造厂商所建议的条件。所举实施例是为了更好地对本发明的内容进行说明，但并不是本发明的内容仅限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
实施例1质粒构建
设计如图1a和1b所示的CAR结构(结构中胞内信号包含胞内共刺激结构域和胞内活化信号)，VL(ScFv1)-linker1-VL(ScFv2)-linker2-VH(ScFv2)-linker1-VH(ScFv1)或VH(ScFv1)-linker1-VL(ScFv2)-linker-VH(ScFv2)-linker1-VL(ScFv1)或VL(ScFv1)-linker1-VH(ScFv2)-linker-VL(ScFv2)-linker1-VH(ScFv1)结构。利用靶向CD19、CD123和CD19、CD22双靶点CAR进行验证，分别设计编码12：抗CD19ScFv轻链-Linker1-抗CD123ScFv轻链-Linker2-抗CD123ScFv重链-Linker1-抗CD19ScFv重链，核苷酸序列如SEQ ID NO:3，氨基酸序列如SEQ ID NO:25所示；编码13：抗CD123ScFv轻链-Linker1-抗CD19ScFv轻链-Linker2-抗CD19ScFv重链-Linker1-抗CD123ScFv重链，核苷酸序列如SEQ ID NO:4，氨基酸序列如SEQ ID NO:26所示；编号19：CD19的ScFv的轻链可变区-Linker1-抗CD22的重链可变区-Linker2-抗CD22的轻链可变区-Linker1-抗CD19的ScFv的重链可变区，核苷酸序列如SEQ ID NO:5，氨基酸序列如SEQ ID NO:27所示；编号20：CD22的ScFv的重链可变区-Linker1-抗CD19的轻链可变区-Linker2-抗CD19的重链可变区-Linker1-抗CD22的ScFv的轻链可变区，核苷酸序列如SEQ ID NO:6，氨基酸序列如SEQ ID NO:28所示。编号12、13的结构如图1a1所示，编号19、20的结构如图1b1所示。
表1：CAR结构原件表
CAR结构原件 核苷酸序列 氨基酸序列
抗CD22ScFv轻链 SEQ ID NO:10 SEQ ID NO:22
抗CD22ScFv重链 SEQ ID NO:9 SEQ ID NO:21
抗CD19ScFv轻链 SEQ ID NO:8 SEQ ID NO:19
抗CD19ScFv重链 SEQ ID NO:7 SEQ ID NO:20
抗CD123ScFv轻链 SEQ ID NO:12 SEQ ID NO:23
抗CD123ScFv重链 SEQ ID NO:11 SEQ ID NO:24
Linker1 SEQ ID NO:1 SEQ ID NO:17
Linker2 SEQ ID NO:2 SEQ ID NO:18
CD8铰链 SEQ ID NO:13 SEQ ID NO:29
CD8跨膜结构 SEQ ID NO:14 SEQ ID NO:30
CD137胞内共刺激结构域 SEQ ID NO:15 SEQ ID NO:31
CD3ζ胞内活化信号 SEQ ID NO:16 SEQ ID NO:32
实施例2制备慢病毒及感染T淋巴细胞
本实施例包装慢病毒采用磷酸钙法，具体为：用含10％FBS(w/v)的DMEM培养基培养293T细胞至较佳状态，包装质粒(RRE:REV:2G)和表达质粒按一定比列加入到1.5的离心管中，加入CaCl2和2×HBS，混匀后室温静置后加入到处理好的293T细胞培养液中，3-5h后再次换液至10mL含10％FBS的DMEM培养基，48h或72h后收集细胞上清，纯化病毒，进行滴度测定。
制备的包含双CAR(12和13)慢病毒感染CHO细胞，分别将感染CHO细胞后的CD19CART、CD123CAR-T、双CART12和双CART13采用CD19-FC、CD123-His流式标记试剂标记后检测双CAR结构中靶向CD19 CAR和靶向CD123 CAR的表达，用Protein-L检测总的CAR表达。
制备的包含双CAR(19和20)慢病毒感染CHO细胞，分别将感染CHO细胞后的CD19CAR-T、CD22 CAR-T、双CAR-T(19和20)采用CD19-FC，CD22-His流式标记试剂标记后检测双CAR结构中靶向CD19 CAR和靶向CD22CAR的表达，用Protein-L检测总的CAR表达。
结果如图2a所示：CD19CAR-T仅能识别CD19-FC，CD123CART仅能识别CD123-His，而双CART12和13可以识别CD19-FC和CD123-His双靶点，并且CAR阳性率大于50％。
结果如图2b所示：CD19 CAR-T仅能识别CD19-FC，CD22 CART仅能识别CD22-His，而双CAR-T(19和20)可以识别CD19-FC和CD22-His双靶点，并且CAR阳性率大于50％。
利用梯度离心法进行淋巴细胞分离；离心后，取第二层白色淋巴细胞层，生理盐水洗涤，加入含有10％FBS的RPMI 1640完全培养基培养，获得人PBMC细胞。获得的PBMC细胞经抗CD3、CD28单克隆抗体活化24h后，按一定的感染复数(MOI)感染已活化的PBMC，在病毒感染的第12天检测CAR-T的阳性率，检测方法为流式检测，抗体为：Protein-L-PE，Protein-L可识别抗体轻链，CAR抗原识别区的ScFv序列的轻链可被Protein-L识别，因此利用Protein-L可检测CAR阳性率和CAR表达强度。
结果如图3a所示：在PBMC细胞CAR表达效率大于40％。
结果如图3b所示：在PBMC细胞CAR表达效率大于20％。
实施例3靶细胞制备和靶抗原检测
(1)CD19和CD123靶抗原检测
培养的靶细胞Raji(人淋巴瘤细胞)、Thp-1(单核巨噬细胞)、Nalm-6(人B淋巴白血病细胞)通过慢病毒感染Luc-GFP病毒制备为带有Luc标记的靶细胞，并采用抗CD19抗体和抗CD123抗体检测靶细胞表面抗原表达。结果如图4a所示：Raji为仅CD19阳性细胞，Thp-1为仅CD123阳性细胞，Nalm-6为CD19和CD123双阳性细胞。
(2)CD19和CD22靶抗原检测
以K562-Luc为模式细胞分别构建外源高表达CD19、CD22、共表达CD19和CD22的靶细胞，并采用抗CD19抗体和抗CD22抗体检测靶细胞表面抗原表达。结果见图4b所示：K562-CD19为仅CD19阳性细胞，K562-CD22为仅CD22阳性细胞，K562-CD19-CD22为CD19和CD22双阳性细胞。
实施例4双靶点CAR-T有效性验证
分别以CD19单阳性的Raji细胞、CD123单阳性的Thp-1细胞，CD19和CD123双阳性的Nalm-6细胞或以CD19单阳性的K562-CD19细胞、CD22单阳性的K562-CD22细胞，CD19和CD22双阳性的K562-CD19-CD22细胞作为靶细胞分别验证编号为12、编号13、编号19和编号20的双靶点CAR-T有效性功能，证明双靶点CAR结构的有效性。将效应细胞(CAR-T细胞)按照一定的效靶比铺于靶细胞中，使用Luciferase Assay System(Promega Cat.#E2520)试剂盒提供的标准方法检测杀伤效果，杀伤率用下列公式计算：
杀伤结果如图5a所示：设计的双CAR结构(12和13)表达的CAR-T细胞不仅对CD19阳性的Raji细胞可以进行有效杀伤，也可以对CD123阳性的Thp-1细胞进行杀伤，还可以对CD19和CD123双阳性的Nalm-6进行杀伤，并且杀伤效果不亚于单靶点CART对单靶点肿瘤细胞的杀伤。
杀伤结果如图5b所示：设计的双CAR结构(19和20)表达的CAR-T细胞不仅对CD19靶点有杀伤效果还对CD22靶点同样具有杀伤效果，还可以对CD19和CD22双阳性的K562-CD19-CD22细胞进行杀伤，并且杀伤效果不亚于单靶点CART对单靶点肿瘤细胞的杀伤。
实施例5双靶点CAR-T细胞在动物模型中的抗肿瘤效果验证
本实施例体内验证使用小鼠为NOD.Cg-PrkdcscidII2rgtm1Sug/JicCrl，简称NOG小鼠，由日本实验动物研究所(CIEA)的Mamoru Ito培育而成，为国际上CAR-T体内相关成瘤实验最常见品系。
(1)CD19和CD123双靶点CAR-T细胞在动物模型中的抗肿瘤效果验证
使用CD19阳性的K562-CD19细胞和CD123阳性的K562-CD123细胞采用1:1混合的方式进行尾静脉回输，构建双靶点小鼠动物模型。图6a为靶细胞K562-CD19：K562-CD123＝1:1混合后表型检测结果，混合后的靶细胞分别具有CD19和CD123的表达。
选择6-8周鼠龄雌性NOD/SCID小鼠，标记耳号后，尾静脉注射K562-CD19：K562-CD123＝1:1混合细胞1×106/只细胞量。成瘤第3天测量小鼠肿瘤荧光强度，根据肿瘤体积随机分为生理盐水组、Control T组、CD19 CAR-T组、CD123 CAR-T组以及双CAR 12结构、双CAR 13结构。成瘤第3天向不同分组小鼠尾静脉注射对应的CAR-T细胞3*10^6CAR-T细胞/只；Control T组于第3天回输总数相同的T淋巴细胞，生理盐水组回输对应体积的生理盐水。
结果如图7a所示：与Control T细胞相比，双靶点CAR-T 12和13具有优异的体内杀伤效果，并且双靶点CART(12和13)的杀伤细胞优于CD19或CD123单靶点CART体内效果。
生存曲线如图8a所示：双靶点CART 12和13治疗的小鼠具有更高的存活时间。
(2)CD19和CD22双靶点CAR-T细胞在动物模型中的抗肿瘤效果验证
使用CD19阳性的K562-CD19细胞和CD22阳性的K562-CD22细胞采用1:1混合的方式进行尾静脉回输，构建双靶点小鼠动物模型。图6b为靶细胞K562-CD19：K562-CD22＝1:1混合后表型检测结果，混合后的靶细胞分别具有CD19和CD22的表达。
选择6-8周鼠龄雌性NOD/SCID小鼠，标记耳号后，尾静脉注射K562-CD19：K562-CD22＝1:1混合细胞1×106/只细胞量。成瘤第3天测量小鼠肿瘤荧光强度，根据肿瘤体积随机分为生理盐水组、Control T组、CD19 CAR-T组、CD22 CAR-T组以及双CAR(19)结构、双CAR(20)结构。成瘤第3天向不同分组小鼠尾静脉注射对应的CAR-T细胞3*106CAR-T细胞/只；Control T组于第3天回输总数相同的T淋巴细胞，生理盐水组回输对应体积的生理盐水。
结果如图7b所示：与Control T细胞相比，双靶点CAR-T(19和20)具有优异的体内杀伤效果，并且双靶点CAR-T(19和20)的杀伤细胞优于CD19或CD22单靶点CAR-T体内效果。
生存曲线如图8b所示：双靶点CAR-T(19和20)治疗的小鼠具有更高的存活时间。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 重庆精准生物科技有限公司
<120> 优化的连接肽组合及其应用
<130> 2020318
<160> 32
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> DNA
<213> Artificial Sequence
<400> 1
gggggtggag gctct 15
<210> 2
<211> 54
<212> DNA
<213> Artificial Sequence
<400> 2
ggaagcacct ccggatctgg caagcctgga tccggagagg gctctacaaa ggga 54
<210> 3
<211> 2172
<212> DNA
<213> Artificial Sequence
<400> 3
atggcactgc cagtgaccgc cctgctgctg cctctggccc tgctgctgca cgcagcacgc 60
ccagacatcc agatgacaca gtcccccagc tccctgtctg ccagcgtggg cgaccgggtg 120
accatcacat gcagagcctc tcaggatatc agcaagtatc tgaactggta ccagcagaag 180
ccaggcaagg cccccaggct gctgatctat cacacctccc gcctgcactc tggagtgcca 240
agccggttct ccggatctgg aagcggaacc gactacaccc tgacaatctc tagcctgcag 300
cctgaggatt tcgccacata ctattgccag cagggcaata ccctgccata tacatttggc 360
ggaggaacca ggctggagat caaggggggt ggaggctctg acatccagat gacacagagc 420
ccaagctccc tgtctgccag cccaggcgac agggtgacca tcacatgcag agcctccaag 480
tctatcagca aggatctggc ctggtaccag gagaagcctg gcaagaccaa caagctgctg 540
atctattccg gctctacact gcagtctgga gtgccaagcc gcttcagcgg atccggatct 600
ggaaccgact ttaccctgac aatctctagc ctgcagccag aggatttcgc cacatactat 660
tgccagcagc acaataagta cccctatacc tttggcggcg gcacaaagct ggagatcaag 720
ggaagcacct ccggatctgg caagcctgga tccggagagg gctctacaaa gggacaggtg 780
cagctggtgc agcctggagc agaggtgaag aagccaggag ccagcgtgaa ggtgtcctgt 840
aaggcctctg gctacacctt cacaagctat tggatgaact gggtgcggca ggcaccagga 900
cagggactgg agtggatggg cagaatcgac ccttacgatt ccgagaccca ctataatcag 960
aagtttaagg accgggtgac catcacagcc gataagagca cctccacagc ctacatggag 1020
ctgtcctctc tgaggtccga ggataccgcc gtgtactatt gtgccagagg caactgggac 1080
gattattggg gccagggcac cacactgacc gtgagctccg ggggtggagg ctctcaggtg 1140
cagctgcagg agtccggacc tggactggtg aagccaagcc agacactgtc cctgacctgt 1200
acagtgagcg gcgtgtccct gcctgattac ggcgtgtcct ggatcagaca gccacctggc 1260
aaggccctgg agtggctggg cgtgatctgg ggctctgaga ccacatacta ttccacctct 1320
ctgaagacca ggctgacaat ctctaaggac aacagcaaga atcaggtggt gctgaccatg 1380
acaaacatgg accctgtgga taccgccaca tactattgtg ccaagcacta ctattacggc 1440
ggcagctatg ccatggatta ctggggccag ggctcctctg tgaccgtgag ctccgtcgag 1500
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 1560
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 1620
gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 1680
ctgtcactgg ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc 1740
aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1800
tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 1860
gcccccgcgt acaagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1920
gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1980
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 2040
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 2100
taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 2160
ccccctcgct aa 2172
<210> 4
<211> 2172
<212> DNA
<213> Artificial Sequence
<400> 4
atggcactgc cagtgaccgc cctgctgctg cctctggccc tgctgctgca cgcagcaagg 60
ccagacatcc agatgacaca gagcccaagc tccctgtctg ccagcccagg cgacagggtg 120
accatcacat gcagagcctc caagtctatc agcaaggatc tggcctggta ccaggagaag 180
cctggcaaga ccaacaagct gctgatctat tccggctcta cactgcagtc tggagtgcca 240
agccgcttca gcggatccgg atctggaacc gactttaccc tgacaatctc tagcctgcag 300
ccagaggatt tcgccacata ctattgccag cagcacaata agtaccccta tacctttggc 360
ggcggcacaa agctggagat caaggggggt ggaggctctg acatccagat gacacagtcc 420
cccagctccc tgtctgccag cgtgggcgac cgggtgacca tcacatgcag agcctctcag 480
gatatcagca agtatctgaa ctggtaccag cagaagccag gcaaggcccc caggctgctg 540
atctatcaca cctcccgcct gcactctgga gtgccaagcc ggttctccgg atctggaagc 600
ggaaccgact acaccctgac aatctctagc ctgcagcctg aggatttcgc cacatactat 660
tgccagcagg gcaataccct gccatataca tttggcggag gaaccaggct ggagatcaag 720
ggatccacat ctggaagcgg caagccagga tccggagagg gatctaccaa gggacaggtg 780
cagctgcagg agtccggacc tggactggtg aagccaagcc agacactgtc cctgacctgt 840
acagtgagcg gcgtgtccct gcctgattac ggcgtgtcct ggatcagaca gccacctggc 900
aaggccctgg agtggctggg cgtgatctgg ggctctgaga ccacatacta ttccacctct 960
ctgaagacca ggctgacaat ctctaaggac aacagcaaga atcaggtggt gctgaccatg 1020
acaaacatgg accctgtgga taccgccaca tactattgtg ccaagcacta ctattacggc 1080
ggcagctatg ccatggatta ctggggccag ggctcctctg tgaccgtgag ctccgggggt 1140
ggaggctctc aggtgcagct ggtgcagcct ggagcagagg tgaagaagcc aggagccagc 1200
gtgaaggtgt cctgtaaggc ctctggctac accttcacaa gctattggat gaactgggtg 1260
cggcaggcac caggacaggg actggagtgg atgggcagaa tcgaccctta cgattccgag 1320
acccactata atcagaagtt taaggaccgg gtgaccatca cagccgataa gagcacctcc 1380
acagcctaca tggagctgtc ctctctgagg tccgaggata ccgccgtgta ctattgtgcc 1440
agaggcaact gggacgatta ttggggccag ggcaccacac tgaccgtgag ctccgtcgag 1500
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 1560
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 1620
gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 1680
ctgtcactgg ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc 1740
aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1800
tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 1860
gcccccgcgt acaagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1920
gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1980
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 2040
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 2100
taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 2160
ccccctcgct aa 2172
<210> 5
<211> 2211
<212> DNA
<213> Artificial Sequence
<400> 5
atggcactgc cagtgaccgc cctgctgctg cctctggccc tgctgctgca cgcagcacgc 60
ccagacatcc agatgacaca gtcccccagc tccctgtctg ccagcgtggg cgaccgggtg 120
accatcacat gcagagcctc tcaggatatc agcaagtatc tgaactggta ccagcagaag 180
ccaggcaagg cccccaggct gctgatctat cacacctccc gcctgcactc tggagtgcca 240
agccggttct ccggatctgg aagcggaacc gactacaccc tgacaatctc tagcctgcag 300
cctgaggatt tcgccacata ctattgccag cagggcaata ccctgccata tacatttggc 360
ggaggaacca ggctggagat caaggaattc gggggtggag gctctcaggt gcagctgcag 420
cagagcggac caggactggt gaagccatcc cagaccctgt ctctgacatg cgccatctcc 480
ggcgattctg tgagctccaa cagcgccgcc tggaattgga tccggcagtc ccccagccgg 540
ggcctggagt ggctgggacg gacatactat agatccaagt ggtacaacga ttatgccgtg 600
agcgtgaagt cccggatcac catcaacccc gacacatcta agaatcagtt cagcctgcag 660
ctgaacagcg tgacccctga ggacacagcc gtgtactatt gtgccagaga ggtgaccggc 720
gacctggagg atgcctttga catctggggc cagggcacca tggtgacagt gtctagcggc 780
agcacatccg gatctggcaa gccaggaagc ggagagggct ccaccaaggg cgatatccag 840
atgacacagt ccccctcctc tctgagcgcc tccgtgggcg acagggtgac catcacatgc 900
cgcgcctctc agaccatctg gagctacctg aactggtatc agcagaggcc aggcaaggca 960
cctaatctgc tgatctacgc agccagctcc ctgcagtccg gagtgccttc taggttcagc 1020
ggaaggggat ctggaaccga cttcaccctg acaatctcta gcctgcaggc cgaggacttc 1080
gccacatact attgtcagca gtcttatagc atcccacaga cctttggcca gggcacaaag 1140
ctggagatca agtctagagg gggtggaggc tctcaggtgc agctgcagga gtccggacct 1200
ggactggtga agccaagcca gacactgtcc ctgacctgta cagtgagcgg cgtgtccctg 1260
cctgattacg gcgtgtcctg gatcagacag ccacctggca aggccctgga gtggctgggc 1320
gtgatctggg gctctgagac cacatactat tccacctctc tgaagaccag gctgacaatc 1380
tctaaggaca acagcaagaa tcaggtggtg ctgaccatga caaacatgga ccctgtggat 1440
accgccacat actattgtgc caagcactac tattacggcg gcagctatgc catggattac 1500
tggggccagg gctcctctgt gaccgtgagc tccctcgaga ccacgacgcc agcgccgcga 1560
ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 1620
cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac 1680
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 1740
tactgcaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 1800
gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1860
ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 1920
cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1980
aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 2040
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 2100
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 2160
accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta a 2211
<210> 6
<211> 2211
<212> DNA
<213> Artificial Sequence
<400> 6
atggcactgc ctgtgaccgc cctgctgctg ccactggccc tgctgctgca cgcagcaagg 60
ccacaggtgc agctgcagca gagcggacca ggactggtga agccatccca gaccctgtct 120
ctgacatgcg ccatctccgg cgattctgtg agctccaaca gcgccgcctg gaattggatc 180
cggcagtccc ccagccgggg cctggagtgg ctgggacgga catactatag atccaagtgg 240
tacaacgatt atgccgtgag cgtgaagtcc cggatcacca tcaaccccga cacatctaag 300
aatcagttca gcctgcagct gaacagcgtg acccctgagg acacagccgt gtactattgt 360
gccagagagg tgaccggcga cctggaggat gcctttgaca tctggggcca gggcaccatg 420
gtgacagtgt ctagcgaatt cgggggtgga ggctctgaca tccagatgac acagtccccc 480
agctccctgt ctgccagcgt gggcgaccgg gtgaccatca catgcagagc ctctcaggat 540
atcagcaagt atctgaactg gtaccagcag aagccaggca aggcccccag gctgctgatc 600
tatcacacct cccgcctgca ctctggagtg ccaagccggt tctccggatc tggaagcgga 660
accgactaca ccctgacaat ctctagcctg cagcctgagg atttcgccac atactattgc 720
cagcagggca ataccctgcc atatacattt ggcggaggaa ccaggctgga gatcaaggga 780
tccacatctg gaagcggcaa gccaggatcc ggagagggat ctaccaaggg acaggtgcag 840
ctgcaggagt ccggacctgg actggtgaag ccaagccaga cactgtccct gacctgtaca 900
gtgagcggcg tgtccctgcc tgattacggc gtgtcctgga tcagacagcc acctggcaag 960
gccctggagt ggctgggcgt gatctggggc tctgagacca catactattc cacctctctg 1020
aagaccaggc tgacaatctc taaggacaac agcaagaatc aggtggtgct gaccatgaca 1080
aacatggacc ctgtggatac cgccacatac tattgtgcca agcactacta ttacggcggc 1140
agctatgcca tggattactg gggccagggc tcctctgtga ccgtgagctc ctctagaggg 1200
ggtggaggct ctgatatcca gatgacacag tccccctcct ctctgagcgc ctccgtgggc 1260
gacagggtga ccatcacatg ccgcgcctct cagaccatct ggagctacct gaactggtat 1320
cagcagaggc caggcaaggc acctaatctg ctgatctacg cagccagctc cctgcagtcc 1380
ggagtgcctt ctaggttcag cggaagggga tctggaaccg acttcaccct gacaatctct 1440
agcctgcagg ccgaggactt cgccacatac tattgtcagc agtcttatag catcccacag 1500
acctttggcc agggcacaaa gctggagatc aagctcgaga ccacgacgcc agcgccgcga 1560
ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 1620
cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac 1680
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 1740
tactgcaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 1800
gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1860
ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 1920
cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1980
aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 2040
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 2100
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 2160
accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta a 2211
<210> 7
<211> 360
<212> DNA
<213> Artificial Sequence
<400> 7
caggtgcagc tgcaggagtc cggacctgga ctggtgaagc caagccagac actgtccctg 60
acctgtacag tgagcggcgt gtccctgcct gattacggcg tgtcctggat cagacagcca 120
cctggcaagg ccctggagtg gctgggcgtg atctggggct ctgagaccac atactattcc 180
acctctctga agaccaggct gacaatctct aaggacaaca gcaagaatca ggtggtgctg 240
accatgacaa acatggaccc tgtggatacc gccacatact attgtgccaa gcactactat 300
tacggcggca gctatgccat ggattactgg ggccagggct cctctgtgac cgtgagctcc 360
<210> 8
<211> 321
<212> DNA
<213> Artificial Sequence
<400> 8
gacatccaga tgacacagtc ccccagctcc ctgtctgcca gcgtgggcga ccgggtgacc 60
atcacatgca gagcctctca ggatatcagc aagtatctga actggtacca gcagaagcca 120
ggcaaggccc ccaggctgct gatctatcac acctcccgcc tgcactctgg agtgccaagc 180
cggttctccg gatctggaag cggaaccgac tacaccctga caatctctag cctgcagcct 240
gaggatttcg ccacatacta ttgccagcag ggcaataccc tgccatatac atttggcgga 300
ggaaccaggc tggagatcaa g 321
<210> 9
<211> 372
<212> DNA
<213> Artificial Sequence
<400> 9
caggtgcagc tgcagcagag cggaccagga ctggtgaagc catcccagac cctgtctctg 60
acatgcgcca tctccggcga ttctgtgagc tccaacagcg ccgcctggaa ttggatccgg 120
cagtccccca gccggggcct ggagtggctg ggacggacat actatagatc caagtggtac 180
aacgattatg ccgtgagcgt gaagtcccgg atcaccatca accccgacac atctaagaat 240
cagttcagcc tgcagctgaa cagcgtgacc cctgaggaca cagccgtgta ctattgtgcc 300
agagaggtga ccggcgacct ggaggatgcc tttgacatct ggggccaggg caccatggtg 360
acagtgtcta gc 372
<210> 10
<211> 321
<212> DNA
<213> Artificial Sequence
<400> 10
gatatccaga tgacacagtc cccctcctct ctgagcgcct ccgtgggcga cagggtgacc 60
atcacatgcc gcgcctctca gaccatctgg agctacctga actggtatca gcagaggcca 120
ggcaaggcac ctaatctgct gatctacgca gccagctccc tgcagtccgg agtgccttct 180
aggttcagcg gaaggggatc tggaaccgac ttcaccctga caatctctag cctgcaggcc 240
gaggacttcg ccacatacta ttgtcagcag tcttatagca tcccacagac ctttggccag 300
ggcacaaagc tggagatcaa g 321
<210> 11
<211> 345
<212> DNA
<213> Artificial Sequence
<400> 11
caggtgcagc tggtgcagcc tggagcagag gtgaagaagc caggagccag cgtgaaggtg 60
tcctgtaagg cctctggcta caccttcaca agctattgga tgaactgggt gcggcaggca 120
ccaggacagg gactggagtg gatgggcaga atcgaccctt acgattccga gacccactat 180
aatcagaagt ttaaggaccg ggtgaccatc acagccgata agagcacctc cacagcctac 240
atggagctgt cctctctgag gtccgaggat accgccgtgt actattgtgc cagaggcaac 300
tgggacgatt attggggcca gggcaccaca ctgaccgtga gctcc 345
<210> 12
<211> 321
<212> DNA
<213> Artificial Sequence
<400> 12
gacatccaga tgacacagag cccaagctcc ctgtctgcca gcccaggcga cagggtgacc 60
atcacatgca gagcctccaa gtctatcagc aaggatctgg cctggtacca ggagaagcct 120
ggcaagacca acaagctgct gatctattcc ggctctacac tgcagtctgg agtgccaagc 180
cgcttcagcg gatccggatc tggaaccgac tttaccctga caatctctag cctgcagcca 240
gaggatttcg ccacatacta ttgccagcag cacaataagt acccctatac ctttggcggc 300
ggcacaaagc tggagatcaa g 321
<210> 13
<211> 135
<212> DNA
<213> Artificial Sequence
<400> 13
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 14
<211> 72
<212> DNA
<213> Artificial Sequence
<400> 14
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 15
<211> 126
<212> DNA
<213> Artificial Sequence
<400> 15
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 16
<211> 336
<212> DNA
<213> Artificial Sequence
<400> 16
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 17
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 17
Gly Gly Gly Gly Ser
1 5
<210> 18
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 18
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
1 5 10 15
Lys Gly
<210> 19
<211> 107
<212> PRT
<213> Artificial Sequence
<400> 19
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys
100 105
<210> 20
<211> 120
<212> PRT
<213> Artificial Sequence
<400> 20
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Thr Ser Leu Lys
50 55 60
Thr Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Val Leu
65 70 75 80
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Ser Ser Val Thr Val Ser Ser
115 120
<210> 21
<211> 124
<212> PRT
<213> Artificial Sequence
<400> 21
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn
20 25 30
Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu Glu Asp Ala Phe Asp
100 105 110
Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 22
<211> 107
<212> PRT
<213> Artificial Sequence
<400> 22
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 Thr Ile Trp Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 23
<211> 107
<212> PRT
<213> Artificial Sequence
<400> 23
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 24
<211> 115
<212> PRT
<213> Artificial Sequence
<400> 24
Gln Val Gln Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser
115
<210> 25
<211> 702
<212> PRT
<213> Artificial Sequence
<400> 25
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 Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly
115 120 125
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
130 135 140
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
145 150 155 160
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
165 170 175
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
180 185 190
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
195 200 205
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly
210 215 220
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
225 230 235 240
Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
245 250 255
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
260 265 270
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile
275 280 285
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Arg
290 295 300
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
305 310 315 320
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
325 330 335
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
340 345 350
Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
355 360 365
Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val
370 375 380
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys
385 390 395 400
Ala Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
405 410 415
Ser Thr Ser Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys
420 425 430
Asn Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala
435 440 445
Thr Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
450 455 460
Asp Tyr Trp Gly Gln Gly Ser Ser Val Thr Val Ser Ser Val Glu Thr
465 470 475 480
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
485 490 495
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
500 505 510
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
515 520 525
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
530 535 540
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
545 550 555 560
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
565 570 575
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
580 585 590
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn
595 600 605
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
610 615 620
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
625 630 635 640
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
645 650 655
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
660 665 670
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
675 680 685
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
690 695 700
<210> 26
<211> 702
<212> PRT
<213> Artificial Sequence
<400> 26
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
115 120 125
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
130 135 140
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile
145 150 155 160
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
165 170 175
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
180 185 190
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
195 200 205
Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Gly Ser Thr Ser Gly
210 215 220
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
225 230 235 240
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser
245 250 255
Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
260 265 270
Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly Val Ile
275 280 285
Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Thr Ser Leu Lys Thr Arg Leu
290 295 300
Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Val Leu Thr Met Thr
305 310 315 320
Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Lys His Tyr
325 330 335
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Ser Ser
340 345 350
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln
355 360 365
Pro Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys
370 375 380
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn Trp Val Arg
385 390 395 400
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Tyr
405 410 415
Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Arg Val Thr Ile
420 425 430
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu
435 440 445
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Asn Trp Asp
450 455 460
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Thr
465 470 475 480
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
485 490 495
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
500 505 510
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
515 520 525
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
530 535 540
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
545 550 555 560
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
565 570 575
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
580 585 590
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn
595 600 605
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
610 615 620
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
625 630 635 640
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
645 650 655
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
660 665 670
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
675 680 685
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
690 695 700
<210> 27
<211> 715
<212> PRT
<213> Artificial Sequence
<400> 27
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 Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Glu Phe Gly Gly Gly
100 105 110
Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro
115 120 125
Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser
130 135 140
Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly
145 150 155 160
Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp
165 170 175
Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser
180 185 190
Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
195 200 205
Ala Val Tyr Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu Glu Asp Ala
210 215 220
Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Ser
225 230 235 240
Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly
245 250 255
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
260 265 270
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr
275 280 285
Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile
290 295 300
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
305 310 315 320
Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala
325 330 335
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln
340 345 350
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Arg Gly Gly Gly
355 360 365
Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
370 375 380
Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro
385 390 395 400
Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
405 410 415
Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Thr Ser
420 425 430
Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val
435 440 445
Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr
450 455 460
Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp
465 470 475 480
Gly Gln Gly Ser Ser Val Thr Val Ser Ser Leu Glu Thr Thr Thr Pro
485 490 495
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
500 505 510
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
515 520 525
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
530 535 540
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
545 550 555 560
Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
565 570 575
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
580 585 590
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
595 600 605
Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr
610 615 620
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
625 630 635 640
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
645 650 655
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
660 665 670
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
675 680 685
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
690 695 700
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
705 710 715
<210> 28
<211> 715
<212> PRT
<213> Artificial Sequence
<400> 28
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn
20 25 30
Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu Glu Asp Ala Phe Asp
100 105 110
Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Glu Phe Gly Gly
115 120 125
Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
130 135 140
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile
145 150 155 160
Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg
165 170 175
Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg
180 185 190
Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser
195 200 205
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr
210 215 220
Leu Pro Tyr Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Gly Ser
225 230 235 240
Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly
245 250 255
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
260 265 270
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
275 280 285
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
290 295 300
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Thr Ser Leu Lys
305 310 315 320
Thr Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Val Leu
325 330 335
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala
340 345 350
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
355 360 365
Gly Ser Ser Val Thr Val Ser Ser Ser Arg Gly Gly Gly Gly Ser Asp
370 375 380
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
385 390 395 400
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr Leu
405 410 415
Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile Tyr
420 425 430
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Arg
435 440 445
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
450 455 460
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln Thr
465 470 475 480
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Leu Glu Thr Thr Thr Pro
485 490 495
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
500 505 510
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
515 520 525
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
530 535 540
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
545 550 555 560
Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
565 570 575
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
580 585 590
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
595 600 605
Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr
610 615 620
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
625 630 635 640
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
645 650 655
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
660 665 670
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
675 680 685
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
690 695 700
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
705 710 715
<210> 29
<211> 47
<212> PRT
<213> Artificial Sequence
<400> 29
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
1 5 10 15
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
20 25 30
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 30
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 30
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 31
<211> 44
<212> PRT
<213> Artificial Sequence
<400> 31
Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
1 5 10 15
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
20 25 30
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
35 40
<210> 32
<211> 111
<212> PRT
<213> Artificial Sequence
<400> 32
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
1 5 10 15
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
20 25 30
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
35 40 45
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
50 55 60
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
65 70 75 80
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
85 90 95
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110

Claims (10)

1.双特异性单链抗体，其特征在于，所述双特异性单链抗体结构为CD22的ScFv的重链可变区-Linker1-抗CD19的轻链可变区-Linker2-抗CD19的重链可变区-Linker1-抗CD22的ScFv的轻链可变区；编码所述linker1的氨基酸序列如SEQ ID NO:17所示，编码所述Linker2结构的氨基酸序列如SEQ ID NO:18所示；所述CD22的ScFv的重链可变区的氨基酸序列如SEQ ID NO:21所示；所述抗CD19的轻链可变区的氨基酸序列如SEQ ID NO:19所示；所述抗CD19的重链可变区的氨基酸序列如SEQ ID NO:20所示；所述抗CD22的ScFv的轻链可变区的氨基酸序列如SEQ ID NO:22所示。
2.包含权利要求1所述的双特异性单链抗体的CAR，所述CAR还包含铰链结构、跨膜结构、胞内共刺激结构域和胞内活化信号，其特征在于，所述CAR的氨基酸序列如SEQ ID NO:28所示。
3.根据权利要求2所述的CAR，其特征在于，所述CAR的核苷酸序列如SEQ ID NO:6所示。
4.包含权利要求2所述的CAR的表达载体。
5.根据权利要求4所述的表达载体，其特征在于，所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体、RNA载体中的任一种。
6.权利要求4所述的表达载体制得的免疫细胞。
7.根据权利要求6所述的免疫细胞，其特征在于，所述免疫细胞为T细胞或B细胞或NK细胞。
8.提升识别靶点效应细胞的能力的方法，其特征在于，所述方法为使用权利要求4所述的表达载体转导免疫细胞，所述靶点为CD19、CD22中的任一种或多种。
9.权利要求2所述的CAR或权利要求4所述的表达载体在制备肿瘤药物中的应用，其特征在于，所述肿瘤选自急性淋巴样白血病、慢性淋巴细胞白血病、慢性髓性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤中的一种或多种；所述肿瘤药物可以抑制Raij、Thp-1、Nalm-6三种肿瘤细胞。
10.根据权利要求9所述的应用，Raij、Thp-1、Nalm-6三种肿瘤细胞作为所述急性淋巴样白血病、慢性淋巴细胞白血病、慢性髓性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤中的一种或多种疾病的体外研究模型。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010107658A2 (en) * 2009-03-16 2010-09-23 Vallera Daniel A Methods and compositions for bi-specific targeting of cd19/cd22
CN106062206A (zh) * 2014-01-15 2016-10-26 酵活有限公司 双特异性cd3和cd19抗原结合构建体
CN107109419A (zh) * 2014-07-21 2017-08-29 诺华股份有限公司 使用cd33嵌合抗原受体治疗癌症
CN107226867A (zh) * 2017-07-25 2017-10-03 重庆精准生物技术有限公司 抗人cd19抗原的嵌合抗原受体及其应用
CN107567461A (zh) * 2014-12-29 2018-01-09 诺华股份有限公司 制备嵌合抗原受体表达细胞的方法
TW201922773A (zh) * 2017-09-29 2019-06-16 美商細胞設計實驗公司 雙特異性抗cd307e及抗bcma嵌合抗原受體之製造方法及用途

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10738116B2 (en) * 2015-03-19 2020-08-11 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Dual specific anti-CD22-anti-CD19 chimeric antigen receptors

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010107658A2 (en) * 2009-03-16 2010-09-23 Vallera Daniel A Methods and compositions for bi-specific targeting of cd19/cd22
CN106062206A (zh) * 2014-01-15 2016-10-26 酵活有限公司 双特异性cd3和cd19抗原结合构建体
CN107109419A (zh) * 2014-07-21 2017-08-29 诺华股份有限公司 使用cd33嵌合抗原受体治疗癌症
CN107567461A (zh) * 2014-12-29 2018-01-09 诺华股份有限公司 制备嵌合抗原受体表达细胞的方法
CN107226867A (zh) * 2017-07-25 2017-10-03 重庆精准生物技术有限公司 抗人cd19抗原的嵌合抗原受体及其应用
TW201922773A (zh) * 2017-09-29 2019-06-16 美商細胞設計實驗公司 雙特異性抗cd307e及抗bcma嵌合抗原受體之製造方法及用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Haploidentical CD19/CD22 bispecific CAR-T cells induced MRD-negative remission in a patient with relapsed and refractory adult B-ALL after haploidentical hematopoietic stem cell transplantation;Jia et al.;《Journal of Hematology & Oncology》;第1-9页 *
Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22;Qin et al.;《Molecular Therapy: Oncolytics》;第127-137页 *

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