CN113493517A - 双特异性单链抗体及其构建的嵌合抗原受体和应用 - Google Patents

双特异性单链抗体及其构建的嵌合抗原受体和应用 Download PDF

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CN113493517A
CN113493517A CN202010254435.4A CN202010254435A CN113493517A CN 113493517 A CN113493517 A CN 113493517A CN 202010254435 A CN202010254435 A CN 202010254435A CN 113493517 A CN113493517 A CN 113493517A
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陈雪娇
黄霞
赵永春
徐艳敏
赵文旭
陈军
齐亚男
单娟娟
张巍
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Chongqing Precision Biotech Co ltd
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Abstract

本发明属于细胞工程领域，具体涉及一种双特异性单链抗体及其构建的嵌合抗原受体及其应用。该单链抗体的重链可变区与轻链可变区之间由linker连接。构建的嵌合抗原受体可以识别两种不同靶点，降低因肿瘤异质性或肿瘤通过降低表面抗原表达而产生的针对CAR‑T的免疫逃逸，转导效率高，减少复发提高CAR‑T临床疗效。

Description

双特异性单链抗体及其构建的嵌合抗原受体和应用
技术领域
本发明属于基因工程领域，具体涉及一种靶向双特异性的嵌合抗原受体及其应用。
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其表面表达的CAR-T靶点抗原产生逃逸(Robbie G.Majzner et al.(2018)Tumor AntigenEscape from CAR T-cell Therapy)，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。如果仅是简单的同时表达两个不同靶点的CAR涉及到不同的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，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，疗效不同等多种因素。
所以，需要一种降低因肿瘤异质性或肿瘤通过降低表面抗原表达而产生的针对CAR-T的免疫逃逸，也能解决CAR结构的转导问题，同时解决CAR-T临床疗效减少复发的双CAR结构。
发明内容
有鉴于此，本发明目的之一在于提供一种双异性单链抗体，该单链抗体能够有效识别两种抗原。
所述单链抗体结构为ScFv1轻链-Linker1-ScFv2轻链-Linker2-ScFv2重链-Linker1-ScFv1重链，其中Linker1结构的氨基酸序列如SEQ ID NO:19或其功能性变体所示，Linker2结构的氨基酸序列如SEQ ID NO:20或其功能性变体所示。
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。
进一步，所述Linker1的核苷酸序列如SEQ ID NO:7或其功能性变体所示，Linker2的核酸序列如SEQ ID NO:8或其功能性变体所示。
进一步，所述ScFv1来源于抗CD19或抗CD123的抗体，所述ScFv2来源于抗CD19或抗CD123的抗体。
进一步，所述单链抗体结构为：抗CD19ScFv轻链-Linker1-抗CD123ScFv轻链-Linker2-抗CD123ScFv重链-Linker1-抗CD19ScFv重链或抗CD123ScFv轻链-Linker1-抗CD19ScFv轻链-Linker2-抗CD19ScFv重链-Linker1-抗CD123ScFv重链。
进一步，所述CD19轻链可变区氨基酸序列如SEQ ID NO:15所示，CD19重链可变区氨基酸序列如SEQ ID NO:16所示；所述CD123轻链可变区氨基酸序列如SEQ ID NO:17所示，所述CD123重链可变区氨基酸序列如SEQ ID NO:18所示。
进一步，所述CD19轻链可变区包含如SEQ ID NO:4所示的核苷酸序列，CD19重链可变区包含如SEQ ID NO:3所示的核苷酸序列；所述CD123轻链可变区包含如SEQ ID NO:6所示的核苷酸序列，所述CD123重链可变区包含如SEQ ID NO:5所示的核苷酸序列。
进一步，所述单链抗体核苷酸如SEQ ID NO:13所示，氨基酸如SEQ ID NO:21所示，或者核苷酸如SEQ ID NO:14所示，氨基酸序列如SEQ ID NO:22所示。
本发明目的之二在于提供一种包含前述双特异性单链抗体的CAR，该CAR可以有效识别两种抗原。
进一步，所述CAR所述嵌合抗原受体铰链区来源于：IgG、CD8、CD7、CD4中一种或多种；嵌合抗原受体跨膜区来源于：CD8、CD28、CD3ε、CD4、CD16、CD137、CD80、CD86中一种或多种；嵌合抗原受体胞内共刺激结构域来源于：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中一种或多种。
进一步，所述CAR可以是第一代CAR，二代CAR，三代CAR或四代CAR。
优选地，所述CAR的包含前述的单链抗体，CD8铰链区，CD8跨膜区、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:9所示，氨基酸序列如SEQ ID NO:23所示；所述CD8跨膜区的核苷酸序列如SEQ ID NO:10所示，氨基酸序列如SEQ ID NO:24；所述CD137胞内共刺激信号核苷酸序列如SEQ ID NO:11所示，氨基酸序列如SEQ ID NO:25所示；所述CD3ζ胞内活化信号核苷酸序列如SEQ ID NO:12所示，氨基酸序列如SEQ ID NO:26所示。
进一步，所述CAR的核苷酸序列如SEQ ID NO:1所示，核苷酸序列如SEQ ID NO:2所示。
进一步，所述CAR的氨基酸序列如SEQ ID NO:27或者如SEQ ID NO:28所示。
本发明目的之三在于提供一种前述CAR修饰的免疫细胞。
进一步，所述免疫细胞为T细胞或B细胞或NK细胞或NKT细胞或DC细胞或巨噬细胞。
优选地，所述免疫细胞为T细胞。
本发明之五在于提供一种包含前述CAR的表达载体。
进一步，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、质粒中的任一种。
本发明目的之六在于提供一种提升识别CD19或/和CD123靶点效应细胞的方法。所述方法为用前述的单链抗体进行识别，所述效应细胞为T细胞或B细胞或NK细胞。
本发明之七在于提供一种前述的单链抗体或前述的CAR或前述的慢病毒载体在制备抗肿瘤药物中的应用。
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。
本发明的有益效果在于：
本发明提供的嵌合抗原受体同时可以识别两种靶点抗原,降低了因肿瘤异质性或肿瘤通过降低表面抗原表达而产生的针对CAR-T的免疫逃逸，提高临床疗效减少复发。
本发明提供的抗原受体是抗原识别结构域串联两种单链抗体，比简单两个不同靶点的双CAR结构的转导效率高，很少产生CAR-T细胞亚群，适用于临床治疗。
附图说明
图1为CAR结构示意图。
图1a为CD19和CD123双CAR结构示意图。
图2为双CAR结构在CHO细胞表达检测结果图。
图3为靶细胞抗原表达结果图。
图4为双CAR结构在T淋巴细胞中表达检测结果图。
图5为双CAR体外杀伤结果图。
图6为小鼠荷瘤靶细胞表型检测结果图。
图7为双CAR体内杀伤结果图。
图8为双CAR回输后荷瘤小鼠生存曲线图。
具体实施方式
本申请文件中，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。
本实施例中，双CAR12、13指的是如图1所示的12、13的CAR结构。
所举实施例是为了更好地对本发明进行说明，但并不是本发明的内容仅局限于所举实施例。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1CAR质粒的构建
设计如图1所示的CAR结构，图1中胞内信号指的是胞内共刺激信号和胞内活化信号。利用靶向CD19和CD123双靶点CAR进行验证，编号12：抗CD19ScFv轻链-Linker1-抗CD123ScFv轻链-Linker2-抗CD123ScFv重链-Linker1-抗CD19ScFv重链-人CD8α来源铰链-人CD8α来源跨膜-CD137胞内共刺激信号-CD3ζ胞内活化信号，编号13：抗CD123ScFv轻链-Linker1-抗CD19ScFv轻链-Linker2-抗CD19ScFv重链-Linker1-抗CD123ScFv重链-人CD8α来源铰链-人CD8α来源跨膜-CD137胞内共刺激信号-CD3ζ胞内活化信号，结构如图1a所示，核酸序列如SEQ ID NO:1或SEQ ID NO:2所示，利用双酶切分别切割并回收片段，基因片段进行连接、转化并挑单克隆，获得CAR载体12和载体13，结构如图1a所示。为了可视化，CAR结构及结构原件的情况如下表1。
表1CAR结构及结构原件
CAR结构原件 核苷酸序列 氨基酸序列
CD19轻链在前CAR SEQ ID NO:1 SEQ ID NO:27
CD123轻链在前CAR SEQ ID NO:2 SEQ ID NO:28
抗CD19ScFv轻链 SEQ ID NO:4 SEQ ID NO:15
抗CD19ScFv重链 SEQ ID NO:3 SEQ ID NO:16
抗CD123ScFv轻链 SEQ ID NO:6 SEQ ID NO:17
抗CD123ScFv重链 SEQ ID NO:5 SEQ ID NO:18
Linker1 SEQ ID NO:7 SEQ ID NO:19
Linker2 SEQ ID NO:8 SEQ ID NO:20
CD8铰链 SEQ ID NO:9 SEQ ID NO:23
CD8跨膜结构 SEQ ID NO:10 SEQ ID NO:24
CD137胞内共刺激结构域 SEQ ID NO:11 SEQ ID NO:25
CD3ζ胞内活化信号 SEQ ID NO:12 SEQ ID NO:26
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后收集细胞上清，纯化病毒，滴度测定，滴度表见下表2。
表2滴度测定表
Figure BDA0002436743310000071
制备的慢病毒感染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结构对CD19靶点和CD123靶点的识别，用Protein-L检测总的CAR表达。
结果如图2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％。
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3所示：在PBMC细胞CAR表达效率大于40％，表达效率高。
实施例3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4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。
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细胞作为靶细胞验证双靶点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
Figure BDA0002436743310000082
LuciferaseAssay 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：
Figure BDA0002436743310000081
杀伤结果如图5所示：我们设计的双CAR结构表达的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。
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
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6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。
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。体内验证使用的成瘤靶细胞为K562-CD19：K562-CD123＝1:1。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、CD19CAR-T组、CD123CAR-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7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8所示：双靶点CART 12和13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> 重庆精准生物技术有限公司
<120> 双特异性单链抗体及其构建的嵌合抗原受体和应用
<130> 2020318
<160> 28
<170> SIPOSequenceListing 1.0
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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> 2
<211> 2172
<212> DNA
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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
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<212> DNA
<213> Artificial Sequence
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caggtgcagc tgcaggagtc cggacctgga ctggtgaagc caagccagac actgtccctg 60
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cctggcaagg ccctggagtg gctgggcgtg atctggggct ctgagaccac atactattcc 180
acctctctga agaccaggct gacaatctct aaggacaaca gcaagaatca ggtggtgctg 240
accatgacaa acatggaccc tgtggatacc gccacatact attgtgccaa gcactactat 300
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<212> DNA
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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> 5
<211> 345
<212> DNA
<213> Artificial Sequence
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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> 6
<211> 321
<212> DNA
<213> Artificial Sequence
<400> 6
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> 7
<211> 15
<212> DNA
<213> Artificial Sequence
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gggggtggag gctct 15
<210> 8
<211> 54
<212> DNA
<213> Artificial Sequence
<400> 8
ggaagcacct ccggatctgg caagcctgga tccggagagg gctctacaaa ggga 54
<210> 9
<211> 135
<212> DNA
<213> Artificial Sequence
<400> 9
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 10
<211> 72
<212> DNA
<213> Artificial Sequence
<400> 10
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 11
<211> 126
<212> DNA
<213> Artificial Sequence
<400> 11
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 12
<211> 336
<212> DNA
<213> Artificial Sequence
<400> 12
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> 13
<211> 1500
<212> DNA
<213> Artificial Sequence
<400> 13
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
<210> 14
<211> 1500
<212> DNA
<213> Artificial Sequence
<400> 14
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
<210> 15
<211> 107
<212> PRT
<213> Artificial Sequence
<400> 15
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> 16
<211> 120
<212> PRT
<213> Artificial Sequence
<400> 16
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> 17
<211> 107
<212> PRT
<213> Artificial Sequence
<400> 17
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> 18
<211> 115
<212> PRT
<213> Artificial Sequence
<400> 18
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> 19
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 19
Gly Gly Gly Gly Ser
1 5
<210> 20
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 20
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
1 5 10 15
Lys Gly
<210> 21
<211> 477
<212> PRT
<213> Artificial Sequence
<400> 21
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
465 470 475
<210> 22
<211> 477
<212> PRT
<213> Artificial Sequence
<400> 22
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
465 470 475
<210> 23
<211> 47
<212> PRT
<213> Artificial Sequence
<400> 23
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> 24
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 24
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> 25
<211> 44
<212> PRT
<213> Artificial Sequence
<400> 25
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> 26
<211> 111
<212> PRT
<213> Artificial Sequence
<400> 26
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
<210> 27
<211> 702
<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 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> 28
<211> 702
<212> PRT
<213> Artificial Sequence
<400> 28
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

Claims (17)

1.双特异性单链抗体，其特征在于，所述单链抗体结构为ScFv1轻链-Linker1-ScFv2轻链-Linker2-ScFv2重链-Linker1-ScFv1重链，其中Linker1结构的氨基酸序列如SEQ IDNO:19或其功能性变体所示，Linker2结构的氨基酸序列如SEQ ID NO:20或其功能性变体所示。
2.权利要求1所述的单链抗体，其特征在于，Linker1的核苷酸序列如SEQ ID NO:7或其功能性变体所示，Linker2的核酸序列如SEQ ID NO:8或其功能性变体所示。
3.根据权利要求1所述的单链抗体，其特征在于，所述ScFv1来源于抗CD19或抗CD123的抗体，所述ScFv2来源于抗CD19或抗CD123的抗体。
4.权利要求3所述的单链抗体，其特征在于，所述单链抗体结构为：抗CD19ScFv轻链-Linker1-抗CD123ScFv轻链-Linker2-抗CD123ScFv重链-Linker1-抗CD19ScFv重链或抗CD123ScFv轻链-Linker1-抗CD19ScFv轻链-Linker2-抗CD19ScFv重链-Linker1-抗CD123ScFv重链。
5.依据权利要求4所述的单链抗体，其特征在于，所述CD19轻链可变区氨基酸序列如SEQ ID NO:15所示，CD19重链可变区氨基酸序列如SEQ ID NO:16所示；所述CD123轻链可变区氨基酸序列如SEQ ID NO:17所示，所述CD123重链可变区氨基酸序列如SEQ ID NO:18所示。
6.根据权利要求4所述的单链抗体，其特征在于，所述CD19轻链可变区包含如SEQ IDNO:4所示的核苷酸序列，CD19重链可变区包含如SEQ ID NO:3所示的核苷酸序列；所述CD123轻链可变区包含如SEQ ID NO:6所示的核苷酸序列，所述CD123重链可变区包含如SEQID NO:5所示的核苷酸序列。
7.根据权利要求1-5所述的单链抗体，其特征在于，所述单链抗体核苷酸如SEQ ID NO:13所示，氨基酸如SEQ ID NO:21所示，或者核苷酸如SEQ ID NO:14所示，氨基酸序列如SEQID NO:22所示。
8.含有根据权利要求1-5所述的单链抗体的CAR，其特征在于，还包含铰链区、跨膜区、胞内共刺激结构域和胞内活化信号。
9.根据权利要求7所述的CAR，其特征在于，所述CAR的包含权利要求1所述的单链抗体，CD8铰链区，CD8跨膜区、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:9所示，氨基酸序列如SEQ ID NO:23所示；所述CD8跨膜区的核苷酸序列如SEQ ID NO:10所示，氨基酸序列如SEQ ID NO:24；所述CD137胞内共刺激信号核苷酸序列如SEQ ID NO:11所示，氨基酸序列如SEQ ID NO:25所示；所述CD3ζ胞内活化信号核苷酸序列如SEQ ID NO:12所示，氨基酸序列如SEQ ID NO:26所示。
10.根据权利要求8所述CAR，其特征在于，所述CAR的核苷酸序列如SEQ ID NO:1所示，核苷酸序列如SEQ ID NO:2所示。
11.权利要求8所示的CAR，其特征在于，所述CAR的氨基酸序列如SEQ ID NO:27或者如SEQ ID NO:28所示。
12.根据权利要求8所述的CAR修饰的免疫细胞。
13.根据权利要求12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。
14.包含权利要求8-11任一项所述的CAR结构的表达载体。
15.根据权利要求14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、质粒中的任一种。
16.提升识别CD19或/和CD123靶点效应细胞的方法，其特征在于，所述方法为用权利要求1所述的单链抗体进行识别，所述效应细胞为T细胞或B细胞或NK细胞。
17.权利要求1所述的单链抗体或权利要求8所述的CAR或权利要求14所述的表达载体在制备抗肿瘤药物中的应用。
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