CN113493521A - 靶向cd19和cd123的双靶点嵌合抗原受体及其应用 - Google Patents
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Abstract
本发明属于免疫治疗技术领域，具体涉及一种可识别CD19和CD123肿瘤相关抗原的双特异性嵌合抗原受体，一种该嵌合抗原受体的表达载体、CAR‑T细胞及其应用。所述双特异性嵌合抗原受体可识别CD19和CD123双靶点，不仅可以有效的表达于T淋巴细胞，而且能够减少肿瘤免疫逃逸的几率，降低CAR‑T治疗后肿瘤复发率。
Description
技术领域
本发明属于免疫治疗技术领域，具体涉及一种双特异性嵌合抗原受体，包含双特异性嵌合抗原受体的载体，一种表达双特异性嵌合抗原受体的CAR-T细胞及其应用。
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，简单的同时表达2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。
发明内容
有鉴于此，本发明的目的之一在于一种靶向CD19和CD123双靶点的双特异性嵌合抗原受体，所述双特异性嵌合抗体可以同时识别CD19和CD123双靶点，可以避免表达CD19肿瘤对CD19 CART的免疫逃逸，对经CD19 CART治疗后复发但CD19为阴性的患者也可以起到治疗作用。
随着CAR-T治疗临床数据的积累，研究者们发现在CAR-T治疗后复发的患者体内CAR-T针对的靶抗原被下调甚至“丢失”，本发明设计可同时识别两个肿瘤相关靶点的双特异性嵌合抗原受体来避免肿瘤表面其中之一抗原丢失，而导致的CAR-T细胞就失去“杀伤目标”。
为实现上述目的，本发明采用以下方案：
所述CAR结构为靶向CD19和CD123双抗原的双特异性嵌合抗原受体，并且其结构为：靶向CD19ScFv-Linker-靶向CD123ScFv-CD8hinge-8TM-41BB-CD3ζ或靶向CD123ScFv-Linker-靶向CD19ScFv-CD8hinge-8TM-41BB-CD3ζ。
进一步，所述的靶向CD19ScFv的氨基酸序列为SEQ ID NO.10，靶向CD123ScFv的氨基酸序列为SEQ ID NO.11。
进一步，所述的靶向CD19ScFv的核苷酸序列为SEQ ID NO.3，靶向CD123ScFv的核苷酸序列为SEQ ID NO.4。
进一步，所述双靶点CAR的Linker核苷酸序列如SEQ ID NO.9所示。
进一步，所述双靶点CAR的Linker氨基酸序列如SEQ ID NO.18所示。
优选的，所述的嵌合抗原受体包含如SEQ ID NO.1或SEQ ID NO.2所述的核苷酸序列。
优选的，所述的嵌合抗原受体连接胞外识别区与跨膜区域的hinge区氨基酸序列如SEQ ID NO.12所示；跨膜区域的氨基酸序列如SEQ ID NO.13所示；胞内共刺激信号域为CD137，氨基酸序列如SEQ ID NO.14所示；胞内信号活化区域为CD3ζ，氨基酸序列如SEQ IDNO.15所示。
优选的，所述的嵌合抗原受体连接胞外识别区与跨膜区域的hinge区核苷酸序列如SEQ ID NO.5所示；跨膜区域的核苷酸序列如SEQ ID NO.6所示；胞内共刺激信号域为CD137，核苷酸序列如SEQ ID NO.7所示；胞内信号活化区域为CD3ζ，核苷酸序列如SEQ IDNO.8所示。
某些实施例中，所述嵌合抗原受体结构中的铰链区序列可以来源于：IgG、CD8、CD7、CD4；所述嵌合抗原受体中跨膜区可以来源于：CD8、CD28、CD3ε、CD4、CD16、CD137、CD80以及CD86；所述嵌合抗原受体中胞内信号区可来源于：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。
本发明的目的之二在于提供一种包含靶向双特异性抗原的嵌合抗原受体的载体。
进一步，所述的嵌合抗原受体的CAR表达载体可以为慢病毒表达载体、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。
优选的，所述的CAR表达载体为慢病毒表达载体。
进一步，所述的CAR表达载体包含如SEQ ID NO:1、SEQ ID NO:2任一项所示的核苷酸序列。
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(visna-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）。
在某些实施例中，CAR表达载体的启动子可以是缺氧诱导调控的启动子。
在某些实施例中，可操作地连接到编码本文所涵盖的CAR的多核苷酸的所述启动子选自由以下组成的群组：WTPGK、截短的PGK启动子、巨细胞病毒立即早期基因启动子(CMV)、延伸因子1α启动子(EF1-α)、磷酸甘油酸激酶-1启动子(PGK)、泛素-C启动子(UBQ-C)、巨细胞病毒增强子/鸡β-肌动蛋白启动子(CAG)、多瘤病毒增强子/单纯疱疹胸苷激酶启动子(MC1)、β肌动蛋白启动子(β-ACT)、猿猴病毒40启动子(SV40)和骨髓增生肉瘤病毒增强子，阴性对照区缺失的、dl587rev引物结合位点取代的(MND)启动子。
本发明的目的之三在于提供一种CAR-T细胞，所述CART细胞可以同时识别CD19和CD123两个肿瘤相关抗原。
进一步，所述的CAR-T细胞可以应用于表达CD19和CD123抗原的肿瘤治疗。
本发明的目的之四在于提供的嵌合抗原受体结构和/或CAR表达载体和/或CAR-T细胞在制备治疗肿瘤的药物中的应用。
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表达细胞的活性的活性剂。活性剂可以是抑制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PD1CAR。
进一步，所述肿瘤共表达CD19和CD123抗原。
本发明的有益效果在于：
1）本发明提供了一种可同时识别CD19和CD123的嵌合抗原受体，可降低肿瘤免疫逃逸的风险，降低CAR-T治疗后肿瘤复发；
2）本发明提供的双特异性嵌合抗原受体，可以有效的表达于T淋巴细胞，增强CAR-T有效性，提高CAR-T细胞针对肿瘤靶细胞的杀伤，能够用于肿瘤的靶向治疗；
3）本发明提供包含嵌合抗原受体的表达载体制备病毒容易，CAR转导效率高，适合工业化生产。
附图说明。
图1：CAR结构图。
图2：双CAR结构在CHO细胞表达检测。
图3：靶细胞抗原表达。
图4：双CAR结构在T淋巴细胞中表达检测。
图5：双CAR体外杀伤结果。
图6：小鼠荷瘤靶细胞表型检测。
图7：双CAR体内杀伤结果。
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1所示的CAR结构，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，获得载体编号分别为10、11。CAR结构原件包含：核苷酸序列如SEQ ID NO:3，氨基酸序列如SEQ ID NO:10的靶向CD19的ScFv；核苷酸序列如SEQ ID NO:4，氨基酸序列如SEQ ID NO:11的靶向CD123的ScFv;核苷酸序列如SEQ ID NO:5，氨基酸序列如SEQ ID NO:12的铰链hinge结构；核苷酸序列如SEQ IDNO:6，氨基酸序列如SEQ ID NO:13的跨膜结构；核苷酸序列如SEQ ID NO:7，氨基酸序列如SEQ ID NO:14的胞内共刺激结构域，核苷酸序列如SEQ ID NO:8，氨基酸序列如SEQ ID NO:15的胞内活化信号；核苷酸序列如SEQ ID NO:9，氨基酸序列如SEQ ID NO:18的连接靶向CD19 ScFv和靶向CD123 ScFv的Linker区；双靶向CAR的氨基酸序列如SEQ ID NO: 16、SEQID NO:17。
CAR结构如图1所示。
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，滴度测定。
滴度表：
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CART10和双CART11采用CD19-FC，CD123-His（Acro，1741d-971F1-NZ）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，结果如图2所示：CD19CAR-T仅能识别CD19-FC，CD123CART仅能识别CD123-His，而双CART19和20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4所示：在PBMC细胞CAR表达效率大于40%，表达效率高。
实施例3：靶细胞制备和靶抗原检测
培养的靶细胞Raji、Thp-1、Nalm-6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-FC，CD123-His（Acro，1741d-971F1-NZ）检测靶细胞表面抗原表达。结果见图3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Steady-Glo® 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：
细胞杀伤率=(1-效应细胞靶细胞共培养孔荧光强度/单独靶细胞孔荧光强度)×100%
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Control T组、CD19 CAR-T组、CD123 CAR-T组以及双CAR 10结构、双CAR 11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*10^6 CAR-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 10和11具有优异的体内杀伤效果。
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> 靶向CD19和CD123的双靶点嵌合抗原受体及其应用
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2256
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
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 caagggatcc acatctggaa gcggcaagcc aggatccgga 420
gagggatcta ccaagggaca ggtgcagctg caggagtccg gacctggact ggtgaagcca 480
agccagacac tgtccctgac ctgtacagtg agcggcgtgt ccctgcctga ttacggcgtg 540
tcctggatca gacagccacc tggcaaggcc ctggagtggc tgggcgtgat ctggggctct 600
gagaccacat actattccac ctctctgaag accaggctga caatctctaa ggacaacagc 660
aagaatcagg tggtgctgac catgacaaac atggaccctg tggataccgc cacatactat 720
tgtgccaagc actactatta cggcggcagc tatgccatgg attactgggg ccagggctcc 780
tctgtgaccg tgagctccgg aggcggcggt tcaggtggtg gcggatctgg cggaggtggt 840
tccggaggtg gaggttcaga catccagatg acacagagcc caagctccct gtctgccagc 900
ccaggcgaca gggtgaccat cacatgcaga gcctccaagt ctatcagcaa ggatctggcc 960
tggtaccagg agaagcctgg caagaccaac aagctgctga tctattccgg ctctacactg 1020
cagtctggag tgccaagccg cttcagcgga tccggatctg gaaccgactt taccctgaca 1080
atctctagcc tgcagccaga ggatttcgcc acatactatt gccagcagca caataagtac 1140
ccctatacct ttggcggcgg cacaaagctg gagatcaagg gaagcacctc cggatctggc 1200
aagcctggat ccggagaggg ctctacaaag ggacaggtgc agctggtgca gcctggagca 1260
gaggtgaaga agccaggagc cagcgtgaag gtgtcctgta aggcctctgg ctacaccttc 1320
acaagctatt ggatgaactg ggtgcggcag gcaccaggac agggactgga gtggatgggc 1380
agaatcgacc cttacgattc cgagacccac tataatcaga agtttaagga ccgggtgacc 1440
atcacagccg ataagagcac ctccacagcc tacatggagc tgtcctctct gaggtccgag 1500
gataccgccg tgtactattg tgccagaggc aactgggacg attattgggg ccagggcacc 1560
acactgaccg tgagctccgt cgagaccacg acgccagcgc cgcgaccacc aacaccggcg 1620
cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 1680
ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 1740
gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 1800
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1860
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1920
gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1980
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 2040
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 2100
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 2160
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 2220
gacgcccttc acatgcaggc cctgccccct cgctaa 2256
<210> 2
<211> 2256
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
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 caagggaagc acctccggat ctggcaagcc tggatccgga 420
gagggctcta caaagggaca ggtgcagctg gtgcagcctg gagcagaggt gaagaagcca 480
ggagccagcg tgaaggtgtc ctgtaaggcc tctggctaca ccttcacaag ctattggatg 540
aactgggtgc ggcaggcacc aggacaggga ctggagtgga tgggcagaat cgacccttac 600
gattccgaga cccactataa tcagaagttt aaggaccggg tgaccatcac agccgataag 660
agcacctcca cagcctacat ggagctgtcc tctctgaggt ccgaggatac cgccgtgtac 720
tattgtgcca gaggcaactg ggacgattat tggggccagg gcaccacact gaccgtgagc 780
tccggaggcg gcggttcagg tggtggcgga tctggcggag gtggttccgg aggtggaggt 840
tcagacatcc agatgacaca gtcccccagc tccctgtctg ccagcgtggg cgaccgggtg 900
accatcacat gcagagcctc tcaggatatc agcaagtatc tgaactggta ccagcagaag 960
ccaggcaagg cccccaggct gctgatctat cacacctccc gcctgcactc tggagtgcca 1020
agccggttct ccggatctgg aagcggaacc gactacaccc tgacaatctc tagcctgcag 1080
cctgaggatt tcgccacata ctattgccag cagggcaata ccctgccata tacatttggc 1140
ggaggaacca ggctggagat caagggatcc acatctggaa gcggcaagcc aggatccgga 1200
gagggatcta ccaagggaca ggtgcagctg caggagtccg gacctggact ggtgaagcca 1260
agccagacac tgtccctgac ctgtacagtg agcggcgtgt ccctgcctga ttacggcgtg 1320
tcctggatca gacagccacc tggcaaggcc ctggagtggc tgggcgtgat ctggggctct 1380
gagaccacat actattccac ctctctgaag accaggctga caatctctaa ggacaacagc 1440
aagaatcagg tggtgctgac catgacaaac atggaccctg tggataccgc cacatactat 1500
tgtgccaagc actactatta cggcggcagc tatgccatgg attactgggg ccagggctcc 1560
tctgtgaccg tgagctccgt cgagaccacg acgccagcgc cgcgaccacc aacaccggcg 1620
cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 1680
ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 1740
gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 1800
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1860
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1920
gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1980
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 2040
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 2100
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 2160
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 2220
gacgcccttc acatgcaggc cctgccccct cgctaa 2256
<210> 3
<211> 735
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
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 gggatccaca tctggaagcg gcaagccagg atccggagag 360
ggatctacca agggacaggt gcagctgcag gagtccggac ctggactggt gaagccaagc 420
cagacactgt ccctgacctg tacagtgagc ggcgtgtccc tgcctgatta cggcgtgtcc 480
tggatcagac agccacctgg caaggccctg gagtggctgg gcgtgatctg gggctctgag 540
accacatact attccacctc tctgaagacc aggctgacaa tctctaagga caacagcaag 600
aatcaggtgg tgctgaccat gacaaacatg gaccctgtgg ataccgccac atactattgt 660
gccaagcact actattacgg cggcagctat gccatggatt actggggcca gggctcctct 720
gtgaccgtga gctcc 735
<210> 4
<211> 720
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
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 gggaagcacc tccggatctg gcaagcctgg atccggagag 360
ggctctacaa agggacaggt gcagctggtg cagcctggag cagaggtgaa gaagccagga 420
gccagcgtga aggtgtcctg taaggcctct ggctacacct tcacaagcta ttggatgaac 480
tgggtgcggc aggcaccagg acagggactg gagtggatgg gcagaatcga cccttacgat 540
tccgagaccc actataatca gaagtttaag gaccgggtga ccatcacagc cgataagagc 600
acctccacag cctacatgga gctgtcctct ctgaggtccg aggataccgc cgtgtactat 660
tgtgccagag gcaactggga cgattattgg ggccagggca ccacactgac cgtgagctcc 720
<210> 5
<211> 135
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 6
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 7
<211> 126
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 8
<211> 336
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
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> 9
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggaggcggcg gttcaggtgg tggcggatct ggcggaggtg gttccggagg tggaggttca 60
<210> 10
<211> 245
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
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 Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser
130 135 140
Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Thr Ser Leu Lys Thr Arg Leu
180 185 190
Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Val Leu Thr Met Thr
195 200 205
Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Ser Ser
225 230 235 240
Val Thr Val Ser Ser
245
<210> 11
<211> 240
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
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 Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
130 135 140
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile
165 170 175
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Arg
180 185 190
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
195 200 205
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
<210> 12
<211> 47
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
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> 13
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
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> 14
<211> 44
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
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> 15
<211> 111
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
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> 16
<211> 730
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
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 Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser
130 135 140
Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Thr Ser Leu Lys Thr Arg Leu
180 185 190
Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Val Leu Thr Met Thr
195 200 205
Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Ser Ser
225 230 235 240
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
245 250 255
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser
260 265 270
Pro Ser Ser Leu Ser Ala Ser Pro Gly Asp Arg Val Thr Ile Thr Cys
275 280 285
Arg Ala Ser Lys Ser Ile Ser Lys Asp Leu Ala Trp Tyr Gln Glu Lys
290 295 300
Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln
305 310 315 320
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
325 330 335
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
340 345 350
Cys Gln Gln His Asn Lys Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys
355 360 365
Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly
370 375 380
Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Val Gln Pro Gly Ala Glu
385 390 395 400
Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
405 410 415
Tyr Thr Phe Thr Ser Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly
420 425 430
Gln Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr
435 440 445
His Tyr Asn Gln Lys Phe Lys Asp Arg Val Thr Ile Thr Ala Asp Lys
450 455 460
Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
465 470 475 480
Thr Ala Val Tyr Tyr Cys Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly
485 490 495
Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Thr Thr Thr Pro Ala
500 505 510
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
515 520 525
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
530 535 540
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
545 550 555 560
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
565 570 575
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
580 585 590
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
595 600 605
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
610 615 620
Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn
625 630 635 640
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
645 650 655
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
660 665 670
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
675 680 685
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
690 695 700
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
705 710 715 720
Ala Leu His Met Gln Ala Leu Pro Pro Arg
725 730
<210> 17
<211> 730
<212> PRT
<213> 人工序列(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 Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
130 135 140
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile
165 170 175
Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Arg
180 185 190
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu
195 200 205
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
210 215 220
Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
225 230 235 240
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
245 250 255
Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
260 265 270
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
275 280 285
Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
290 295 300
Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser
305 310 315 320
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
325 330 335
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn
340 345 350
Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Gly
355 360 365
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
370 375 380
Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
385 390 395 400
Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp
405 410 415
Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp
420 425 430
Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Thr Ser Leu
435 440 445
Lys Thr Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Val
450 455 460
Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys
465 470 475 480
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly
485 490 495
Gln Gly Ser Ser Val Thr Val Ser Ser Val Glu Thr Thr Thr Pro Ala
500 505 510
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
515 520 525
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
530 535 540
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
545 550 555 560
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
565 570 575
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
580 585 590
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
595 600 605
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
610 615 620
Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn
625 630 635 640
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
645 650 655
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
660 665 670
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
675 680 685
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
690 695 700
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
705 710 715 720
Ala Leu His Met Gln Ala Leu Pro Pro Arg
725 730
<210> 18
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
Claims (12)
1.靶向CD19和CD123双抗原的双特异性嵌合抗原受体，其特征在于，所述双特异性嵌合抗原受体结构为：靶向CD19ScFv-Linker-靶向CD123ScFv-CD8hinge-8TM-41BB-CD3ζ或靶向CD123ScFv-Linker-靶向CD19ScFv-CD8hinge-8TM-41BB-CD3ζ。
2.权利要求1所述的嵌合抗原受体，其特征在于，靶向CD19ScFv的氨基酸序列为SEQ IDNO.10，靶向CD123ScFv的氨基酸序列为SEQ ID NO.11。
3.权利要求1所述的嵌合抗原受体，其特征在于，靶向CD19ScFv的核苷酸序列为SEQ IDNO.3，靶向CD123ScFv的核苷酸序列为SEQ ID NO.4。
4.权利要求1所述的嵌合抗原受体，其特征在于，所述Linker核苷酸序列如SEQ IDNO.9所示。
5.权利要求4所述的嵌合抗原受体，其特征在于，所述Linker氨基酸序列如SEQ IDNO.18所示。
6.权利要求1所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体包含如SEQ IDNO.1或SEQ ID NO.2所述的核苷酸序列。
7.权利要求1-5所述的嵌合抗原受体，其特征在于，连接胞外识别区与跨膜区域的hinge区氨基酸序列如SEQ ID NO.12所示；跨膜区域的氨基酸序列如SEQ ID NO.13所示；胞内共刺激信号域为CD137，氨基酸序列如SEQ ID NO.14所示；胞内信号活化区域为CD3ζ，氨基酸序列如SEQ ID NO.15所示。
8.权利要求1-5所述的嵌合抗原受体，其特征在于，连接胞外识别区与跨膜区域的hinge区核苷酸序列如SEQ ID NO.5所示；跨膜区域的核苷酸序列如SEQ ID NO.6所示；胞内共刺激信号域为CD137，核苷酸序列如SEQ ID NO.7所示；胞内信号活化区域为CD3ζ，核苷酸序列如SEQ ID NO.8所示。
9.包含权利要求1-5任一项所述的嵌合抗原受体的CAR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，所述载体包含如SEQ ID NO:1、SEQ ID NO:2任一项所示的核苷酸序列。
10.表达权利要求9所述的嵌合抗原受体的CAR-T细胞。
11.权利要求1所述的CAR结构和/或权利要求9所述的表达载体和/或权利要求10所述的CAR-T细胞在制备治疗肿瘤的药物中的应用。
12.根据权利要求11所述的应用，其特征在于，所述肿瘤共表达CD19和CD123抗原。
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