CN112646033B

CN112646033B - 靶向cd123的嵌合抗原受体及其用途

Info

Publication number: CN112646033B
Application number: CN202011498660.9A
Authority: CN
Inventors: 何霆; 齐菲菲; 鲁薪安; 丁艳萍; 梁梦梦
Original assignee: Shandong Jinsai Biotechnology Co ltd; Beijing Yimiao Shenzhou Pharmaceutical Technology Co ltd
Current assignee: Shandong Jinsai Biotechnology Co ltd; Beijing Yimiao Shenzhou Pharmaceutical Technology Co ltd
Priority date: 2020-12-16
Filing date: 2020-12-16
Publication date: 2022-04-12
Anticipated expiration: 2040-12-16
Also published as: AU2021398871A1; CN112646033A; JP2023554126A; AU2021398871A9; WO2022127401A1; EP4265638A1

Abstract

本发明提供了特异性结合CD123的单链抗体scFv，其包含选自SEQ ID NO：56、57、58、59、60和61的氨基酸序列。还提供了靶向CD123的嵌合抗原受体，其包含选自SEQ ID NO：65、66、67、68、69和70的氨基酸序列。还提供了编码所述scFv或嵌合抗原受体的核酸分子，包含所述核酸分子的载体和细胞，以及所述抗体、嵌合抗原受体、核酸分子、载体或细胞用于制备治疗表达CD123的肿瘤的药物的用途。

Description

靶向CD123的嵌合抗原受体及其用途

技术领域

本发明涉及具有不同抗原识别域的靶向CD123的scFv序列，基于这些scFv序列设计的嵌合抗原受体(CAR)和CAR-T细胞，以及其用于治疗表达CD123的肿瘤、特别是急性髓系白血病的用途。

背景技术

急性髓系白血病(AML)是一种发病率不断上升的恶性肿瘤，主要影响中位年龄为66岁的老年人。美国国家癌症研究所(National Cancer Institute)估计，2018年的新病例将达到19520例，而其中可能会有10670人死于这种疾病[1]。尽管65-74岁的患者在过去连续三十年中，12个月生存率从20％提高到30％，但目前AML的治疗效果仍不令人满意，这些患者的5年生存率＜5％[2]。对于较年轻的AML患者，主要的治疗方法是强化化疗，对于复发风险较高的患者(治愈率35-40％)，采用异基因造血细胞移植(AHCT)，而老年患者的预后仍然很差[2]。复发的AML患者获得第二次缓解的几率小于50％，而在第二次抢救性化疗后，只有16％的患者能实现病情控制[3]。自20世纪70年代引入蒽环类和阿糖胞苷类联合化疗以来，它一直是主要的治疗方法[4，5]。因此，对于此疾病，需要新的治疗策略。

嵌合抗原受体T细胞(CAR-T细胞)的概念首先提出于20世纪80年代后期，其结合了抗体特异性和T细胞杀伤效应，形成过继性免疫效应[6，7]。嵌合抗原受体(CAR)分子结构主要包括抗体单链可变片段(scFv)、胞内信号域及连接scFv与胞内信号域的铰链区和跨膜区。近年来，利用抗CD19和抗CD20嵌合抗原受体T细胞(CAR-T细胞)治疗淋巴样恶性肿瘤(B细胞急性淋巴细胞白血病和非霍奇金淋巴瘤)取得了良好的效果[8]。目前，针对AML治疗的CAR-T靶点，有CD123、CD33、CLL1、FLT3等；其中，靶向CD123的CAR-T研究最多。

CD123是IL-3受体a链(IL-3Ra)。CD123与CD131一起组成高亲和力的IL-3受体，与IL-3结合后，能够促进细胞增殖分化，同时抑制造血细胞凋亡[9，10]。CD123的高表达与信号转导及转录激活因子5(STAT5)信号通路的活化相关。STAT5激活通路参与在多种恶性血液病中[11]。CD123主要在造血***中表达，CD123在正常CD34+CD38-细胞中低表达或不表达[12]。此外，正常情况下在髓系祖细胞、B淋巴祖细胞中CD123有较高表达，而红系祖细胞和多向分化潜能祖细胞中低表达或不表达[13，14]。CD123在60％-90％以上的AML原始细胞中高表达[15-18]。Bras AE等人[19]检测了455例AML患者，其中约55％的患者AML细胞表达CD123，且阳性率达80％以上。

虽然，CAR-T细胞在肿瘤细胞治疗中已取得了较好效果，但是CAR-T的疗效和安全性依然存在较大问题，主要有脱靶效应、细胞因子风暴、神经毒性等[20]。CAR-T细胞的最佳疗效和安全性，取决于多方面因素，其中CAR-T细胞的有效活化具有重要作用。CAR-T细胞的活化程度受CAR分子scFv的亲和力、铰链区长度、共刺激信号域、靶细胞表面抗原密度等多种因素影响。CAR分子的scFv作为与靶细胞表面抗原直接结合的区域，其亲和力对整体CAR-T细胞的活化意义重大。CAR分子scFv亲和力较低时，CAR-T细胞不能得到足够的抗原识别，从而无法达到最佳激活状态；CAR分子scFv亲和力较高时，CAR-T细胞可能会在识别肿瘤细胞的同时也能够识别抗原密度相对较低的正常细胞，产生严重脱靶效应。

因此，为了进一步提高CAR-T细胞的安全性和有效性，保证CAR-T细胞的有效活化，避免脱靶效应，仍需要筛选针对靶点具有适中亲和力的scFv。

发明内容

第一方面，本发明提供了一种特异性结合CD123的抗体，其包括重链可变结构域VH和轻链可变结构域VL，所述重链可变结构域包含SEQ ID NO：1(GYX₁X₂X₃X₄(D)YX₅X₆X₇)的VH-CDR1氨基酸序列，SEQ ID NO：2

(X₈X₉X₁0(X₁₁)X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈NX₁₉X₂₀X₂₁KX₂₂X₂₃X₂₄)的VH-CDR2氨基酸序列和SEQID NO：3

(AX₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁(YD)X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀)的VH-CDR3氨基酸序列；其中X₁选自S、T；X₂选自F、I；X₃选自T、S、M；X₄选自S、D、T、G；X₅选自W、Y、V、A、N；X₆选自M、V、I、W；X₇选自N、H；X₈选自Y、R；X₉选自I、C；X₁₀选自D、N、Y、S；X₁₁选自P、C；X₁₂选自Y、G；X₁₃选自D、N、S；X₁₄选自S、N、D、G；X₁₅选自E、A、G、S；X₁₆选自T、I、S；X₁₇选自H、S、N；X₁₈选自Y、S；X₁₉选自Q、E、P；X₂₀选自K、N、S；X₂₁选自F、L；X₂₂选自D、G、S；X₂₃选自K、R；X₂₄选自A、I；X₂₅选自R、G；X₂₆选自G、S、E；X₂₇选自E、P、R；X₂₈选自G、S；X₂₉选自N、Y、L、W；X₃₀选自W、Y、L；X₃₁选自G、D；X₃₂选自R、E；X₃₃选自Y、S、T；X₃₄选自D、Y、G；X₃₅选自G、Y；X₃₆选自Y、L；X₃₇选自A、G、P；X₃₈选自M、L；X₃₉选自D、A；X₄₀选自Y、C；所述轻链可变结构域包含SEQ ID NO：4(X₄₁SX₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉(QK)X₅₀X₅₁X₅₂X₅₃W)的VL-CDR1氨基酸序列，SEQ ID NO：5(X₅₄SX₅₅X₅₆X₅₇X₅₈X₅₉)的VL-CDR2氨基酸序列和SEQ IDNO：6(X₆₀X₆₁X₆₂X₆₃X₆₄(P)X₆₅TF)的VL-CDR3氨基酸序列；其中X₄₁选自A、S；X₄₂选自Q、K、S；X₄₃选自S、D；X₄₄选自I、V、L；X₄₅选自S、N、D、L；X₄₆选自K、Y、S、D、N；X₄₇选自D、S；X₄₈选自G、S；X₄₉选自D、N；X₅₀选自S、N；X₅₁选自D、Y；X₅₂选自L、I、M；X₅₃选自A、N、H；X₅₄选自G、T、A；X₅₅选自T、N、Q；X₅₆选自L、S、R；X₅₇选自Q、E、A、I、D；X₅₈选自S、P；X₅₉选自G、E；X₆₀选自Q、N；X₆₁选自H、Y、S、F、G；X₆₂选自N、D、H、Y；X₆₃选自K、L、E、R、S；X₆₄选自Y、L、D、S、F、T；X₆₅选自Y、R、W；优选地，所述抗体是scFv。

第二方面，本发明提供了一种靶向CD123的嵌合抗原受体，其包含：第一方面所述的抗体；铰链区；跨膜结构域；共刺激结构域；和CD3ζ胞内信号转导结构域。

第三方面，本发明提供了一种核酸分子，其包含编码第一方面的抗体或第二方面的嵌合抗原受体的核苷酸序列。

第四方面，本发明提供了一种载体，其包含第三方面的核酸分子。

第五方面，本发明提供了一种细胞，其经第四方面的载体转染，从而包含第三方面的核酸分子或第四方面的载体。

第六方面，本发明提供了一种药物组合物，其包含第一方面的抗体、第二方面的嵌合抗原受体、第三方面的核酸分子、第四方面的载体或第五方面的细胞，以及可药用载体。

第七方面，本发明提供了第一方面的抗体、第二方面的嵌合抗原受体、第三方面的核酸分子、第四方面的载体或第五方面的细胞用于制备治疗表达CD123的肿瘤的药物的用途。

本发明通过对靶点CD123的CAR分子的scFv进行亲和力筛选以及CAR-T细胞杀伤效应比较，以获得靶点CD123的最佳scFv序列，使得CAR-T细胞具有更优的安全性和有效性。与现有技术相比，本发明能有效提高靶向CD123的CAR-T细胞对肿瘤细胞系及原代肿瘤细胞的杀伤效率，提高细胞增殖能力。

附图说明

图1表示不同scFv的CD123 CAR分子在T细胞中的转导效率。

图2表示CD123 CAR-T在培养过程中的总细胞增殖情况。

图3表示靶细胞AML原代细胞和THP-1细胞分别刺激下，对CD123 CAR-T细胞分化的影响。

图4表示CD123 CAR-T对CD123阳性细胞系(THP-1)及CD123阴性细胞系(K562)的杀伤效率。

图5表示CD123 CAR-T对AML原代细胞的杀伤效率。

图6表示CD123 CAR-T在AML原代细胞刺激5小时后的细胞因子表达。

具体实施方式

本发明提供了一种特异性结合CD123的抗体，其包括重链可变结构域VH和轻链可变结构域VL，所述重链可变结构域包含VH-CDR1、VH-CDR2和VH-CDR3，所述轻链可变结构域包含VL-CDR1、VL-CDR2和VL-CDR3。

在一个具体实施方案中，所述VH-CDR1选自由SEQ ID NO：8、9、10、11、12组成的组中的氨基酸序列；所述VH-CDR2选自由SEQ ID NO：14、15、16、17、18组成的组中的氨基酸序列；所述VH-CDR3选自由SEQ ID NO：20、21、22、23、24组成的组中的氨基酸序列；所述VL-CDR1选自由SEQ ID NO：26、27、28、29、30组成的组中的氨基酸序列；所述VL-CDR2选自由SEQID NO：32、33、34、35、36组成的组中的氨基酸序列；所述VL-CDR3选自由SEQ ID NO：38、39、40、41、42组成的组中的氨基酸序列。

特别地，本发明的特异性结合CD123的抗体包括：

1)包含如SEQ ID NO：8所示的VH-CDR1、如SEQ ID NO：14所示的VH-CDR2和如SEQID NO：20所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：26所示的VL-CDR1、如SEQID NO：32所示的VL-CDR2和如SEQ ID NO：38所示的VL-CDR3的轻链可变结构域；和/或

2)包含如SEQ ID NO：9所示的VH-CDR1、如SEQ ID NO：15所示的VH-CDR2和如SEQID NO：21所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：27所示的VL-CDR1、如SEQID NO：33所示的VL-CDR2和如SEQ ID NO：39所示的VL-CDR3的轻链可变结构域；和/或

3)包含如SEQ ID NO：10所示的VH-CDR1、如SEQ ID NO：16所示的VH-CDR2和如SEQID NO：22所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：28所示的VL-CDR1、如SEQID NO：34所示的VL-CDR2和如SEQ ID NO：40所示的VL-CDR3的轻链可变结构域；和/或

4)包含如SEQ ID NO：11所示的VH-CDR1、如SEQ ID NO：17所示的VH-CDR2和如SEQID NO：23所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：29所示的VL-CDR1、如SEQID NO：35所示的VL-CDR2和如SEQ ID NO：41所示的VL-CDR3的轻链可变结构域；和/或

5)包含如SEQ ID NO：12所示的VH-CDR1、如SEQ ID NO：18所示的VH-CDR2和如SEQID NO：24所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：30所示的VL-CDR1、如SEQID NO：36所示的VL-CDR2和如SEQ ID NO：42所示的VL-CDR3的轻链可变结构域。

在另一个具体实施方案中，本发明的抗体中的所述重链可变结构域包含选自由SEQ ID NO：44、45、46、47和48组成的组中的氨基酸序列，或包含与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列；和/或所述轻链可变结构域包含选自由SEQID NO：50、51、52、53和54组成的组中的氨基酸序列，或包含与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列。

特别地，本发明提供了一种特异性结合CD123的抗体，其包含：

1)如SEQ ID NO：44所示的重链可变结构域和如SEQ ID NO：50所示的轻链可变结构域；和/或

2)如SEQ ID NO：45所示的重链可变结构域和如SEQ ID NO：51所示的轻链可变结构域；和/或

3)如SEQ ID NO：46所示的重链可变结构域和如SEQ ID NO：52所示的轻链可变结构域；和/或

4)如SEQ ID NO：47所示的重链可变结构域和如SEQ ID NO：53所示的轻链可变结构域；和/或

5)如SEQ ID NO：48所示的重链可变结构域和如SEQ ID NO：54所示的轻链可变结构域；

在另一个具体实施方案中，本发明提供了一种特异性结合CD123的抗体，其包含：选自由SEQ ID NO：56、57、58、59、60和61组成的组中的氨基酸序列，或与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列。

优选地，本发明所述抗体为单链抗体scFv；更优选地，所述单链抗体scFv的重链可变结构域和轻链可变结构域由富含甘氨酸和丝氨酸的接头肽链相连，并且重链可变结构域和轻链可变结构域的顺序可互换。

本发明还提供了一种靶向CD123的嵌合抗原受体，其包含：本发明所述的抗体；铰链区；跨膜结构域；共刺激结构域；和CD3ζ胞内信号转导结构域。

在具体实施方案中，所述共刺激结构域选自CD27、CD28、4-1BB、OX-40、CD30、CD40、PD-1、ICOS、LFA-1、CD-2、CD7、LIGHT、NKG2C、B7-H3或其任意组合；优选地，所述共刺激结构域选自4-1BB或CD28，所述4-1BB包含如SEQ ID NO：62所述的氨基酸序列。

在一个具体实施方案中，所述CD3ζ胞内信号转导结构域包含如SEQ ID NO：63所述的氨基酸序列。

在一个具体实施方案中，所述铰链区和跨膜结构域选自IgG1、IgG4、CD8α、CD28、IL-2受体、IL-7受体、IL-11受体、PD-1或CD34的铰链区和跨膜结构域。

优选地，所述的嵌合抗原受体包含选自由SEQ ID NO：65、66、67、68、69和70组成的组中的氨基酸序列，或与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列。

本发明还提供了一种核酸分子，其包含编码本发明的抗体或嵌合抗原受体的核苷酸序列。

本发明还提供了一种载体，其包含本发明的核酸分子；优选地，所述载体是病毒载体；更优选地，所述载体是慢病毒载体。

本发明还提供了一种细胞，其经本发明的载体转染，从而包含本发明的核酸分子或载体；优选地，所述细胞是T细胞。

本发明还提供了本发明的抗体、嵌合抗原受体、核酸分子、载体或细胞用于制备治疗表达CD123的肿瘤的药物的用途；优选地，所述表达CD123的肿瘤是急性髓系白血病(AML)。

除非特别指明，本文所述技术方案可以任意组合。

本发明进一步通过以下实施例来说明。

实施例1：CD123 CAR分子设计及CAR-T制备

本实施例以设计具有不同抗原识别域的CD123 CAR分子，及CAR-T细胞制备为例。

抗原识别域设计原则：CAR-T-1为对照组，其CD123的scFv序列来自宾夕法尼亚大学报道[21]，在本文中命名为scFv-01。CAR-T-2～7为改造组，其scFv来源于小鼠免疫CD123胞外区融合蛋白所得单克隆抗体序列，在本文中命名为scFv-02-07。

将不同抗原识别域的CD123 scFv的核苷酸序列通过基因合成的方法合成，然后通过同源重组方法与CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ胞内区序列连接形成完整的CAR分子，即CAR 1-7。将此CAR核酸片段通过限制性内切酶酶切和连接酶连接的方式，构建到慢病毒转移质粒pLenti6.3/V5(Thermo Fisher，Waltham，MA，USA)中。将慢病毒包装质粒pLP/VSVG，pLP1/MDK，pLP2/RSK(Thermo Fisher，Waltham，MA，USA)与上述获得的转移质粒，用Lipofectamine 3000(Thermo Fisher，Waltham，MA，USA)转染至HEK293T细胞，48小时后收集培养基，300g离心去除细胞碎片后，用超速离心机25000rpm离心3小时。将沉淀用1mL生理盐水溶解，即为所需的慢病毒载体。

从健康志愿者的外周血单个核细胞中，使用CD3/CD28 beads(Thermo Fisher，Cat#40203D)，分离出T细胞。将分离纯化后的T细胞以1.0×10⁶个细胞/mL，接入含有500IU/mL IL-2(山东金泰生物工程有限公司，中国)的X-VIVO 15培养基(Lonza，Switzerland)中培养。在培养48小时后，用带有CD123 CAR分子的慢病毒载体以MOI＝0.5感染T细胞，以获得CAR-T-1-7。慢病毒感染24小时后，进行细胞换液，并接入新鲜培养体系X-VIVO 15加500IU/mL IL-2中继续培养。在慢病毒感染5天后，使用移液枪反复吹打培养体系中的细胞并收集至离心管，置于磁力架上去除CD3/CD28 beads。T细胞离心并计数，取部分细胞使用流式细胞仪(NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA)检测各组细胞的CAR分子转导效率。

图1所示为T细胞感染慢病毒5天、7天、9天、12天后，各组细胞的CAR分子转导效率。与对照组CAR-T-1相比，CAR-T-2、CAR-T-3、CAR-T-7组的CAR分子转导效率与其接近，CAR-T-4、CAR-T-5、CAR-T-6组明显高于对照组，说明各组CAR-T细胞的CAR蛋白表达情况均较好。

实施例2：改造后CD123 CAR-T的体外增殖能力

本实施例以检测具有不同抗原识别域的CD123 CAR-T的增殖能力为例。

在CD123 CAR-T细胞培养过程中，以慢病毒感染T细胞为培养第二天，之后每隔2至3天，收集CAR-T细胞并离心，加1-2毫升的培养体系重悬，取10微升的细胞稀释一定倍数后，用台盼蓝(Solarbio，Cat#C0040)进行染色，倒置显微镜(Ts2-FL，Nikon，日本)下分别计活细胞总数与死细胞总数，计算出各组细胞的增殖情况。

图2显示，与对照组CAR-T-1相比，具有不同抗原识别域的CAR-T-2和CAR-T-7的总细胞增殖有明显提高。说明CAR-T-2和CAR-T-7的scFv设计能够改善CD123 CAR-T细胞增殖能力。

实施例3：改造后CD123 CAR-T在靶细胞刺激下的细胞分化情况

本实施例以检测具有不同抗原识别域的CD123 CAR-T，分别在两种靶细胞AML原代细胞或THP-1细胞(均表达CD123)的刺激下，其T细胞分化情况为例。

CAR-T细胞培养至第5天时，各组分别取两份5×10⁵个细胞于12孔板中，以效靶比(E：T)＝5：1分别加入靶细胞AML原代细胞或THP-1细胞。在靶细胞刺激6天后，即CAR-T细胞培养至第11天时，各组分别取1×10⁶个细胞并离心，100微升DPBS(HyClone，CAT#SH30028.02)重悬；然后每组CAR-T细胞分别加T细胞分化相关荧光抗体进行标记，使用全光谱流式细胞仪(N7-00008-0A，Cytek Biosciences，Inc.Fremont)进行检测。

图3的结果中，CD45RA+CD62L+细胞群表示初始T细胞群和干性记忆T细胞群的组合，CD45RA+CD62L-细胞群为效应T细胞群。具有不同抗原识别域的CAR-T-2和CAR-T-7的CD45RA+CD62L+细胞群占比高于对照组CAR-T-1，且CD45RA+CD62L-细胞群占比低于对照组CAR-T-1。说明改造后的CD123抗原识别域具有更好的CAR-T细胞持久性。

实施例4：改造后CD123 CAR-T在体外对细胞系的杀伤效率

本实施例以检测具有不同抗原识别域的CD123 CAR-T对表达CD123和不表达CD123的肿瘤细胞系的杀伤效率为例。

分别收集并离心表达CD123的细胞系THP-1，以及不表达CD123的细胞系K562，用1毫升生理盐水(河北天成药业有限公司，中国)重悬，加5微升Calcein-AM(浓度1μg/μL，Cat#C3100MP，ThermoFisher，USA)，轻轻混匀，置于37℃培养箱中孵育30分钟，以标记靶细胞。孵育结束后，使用生理盐水清洗2次，加X-VIVO 15重悬并计数。向48孔细胞培养板(CorningIncorporated，Corning，NY，USA)中，每孔加入1×10⁵个上述标记的靶细胞，并以E：T＝10：1的比例加入各组CAR-T细胞，置于37℃、5％CO₂培养箱中孵育5小时。孵育完成后，加2％浓度的Triton-X-100(Cat#T8787-100ML，Sigma，Germany)裂解阳性对照组，即1×10⁵个上述标记的靶细胞；每孔分别取100微升杀伤体系上清于酶标板中，使用多功能酶标仪(VarioscanLux，ThermoFisher)检测荧光值(激发波长：495nm，发射波长：515nm)。

图4显示，对于CD123阳性细胞系THP-1，具有不同抗原识别域的CD123 CAR-T中CAR-T-3和CAR-T-5的杀伤效率，较对照组CAR-T-1显著升高。同时，各组改造后CD123 CAR-T和对照组CAR-T-1，对不表达CD123的K562细胞系的杀伤效率，与T细胞组一致。说明各组CD123 CAR-T细胞对靶细胞均有一定杀伤效应，并有较好的特异性，且CAR-T-3和CAR-T-5的scFv设计更有利于CAR-T杀伤肿瘤细胞。

实施例5：改造后CD123 CAR-T在体外对AML原代细胞的杀伤效率

本实施例以检测具有不同抗原识别域的CD123 CAR-T对AML原代细胞的杀伤效率为例。

取3个分别为高抗原密度(即AML 1#，CD123表达率为99.87％，MFI为13298)、中抗原密度(即AML 2#，CD123表达率为75.4％，MFI为7518)、低抗原密度(即AML 3#，CD123表达率为94.58％，MFI为5252)的CD123阳性AML患者骨髓血样(抗原密度水平定义如下，高抗原密度：中位荧光强度(Median Fluorescence Intensity，MFI)大于10000；中抗原密度：MFI大于6000小于10000；低抗原密度：MFI小于6000)，将AML患者的骨髓血样本分离单个核细胞，并使用CD34 MicroBeads(Cat#130-100-453，美天旎，德国)分选出CD34阳性AML原代细胞，用1毫升生理盐水重悬，加10微升Calcein-AM(浓度1μg/μL，ThermoFisher，USA)，轻轻混匀，置于37℃培养箱中孵育30分钟，以标记AML原代细胞。孵育结束后，使用生理盐水清洗2次，加X-VIVO 15重悬并计数。向48孔细胞培养板(Corning Incorporated，Corning，NY，USA)中，每孔加入1×10⁵个上述标记的AML原代细胞，并以E∶T＝10∶1的比例，分别加入CD123 CAR-T细胞，置于37℃、5％CO₂培养箱中孵育5小时。孵育完成后，加2％浓度的Triton-X-100裂解阳性对照组，即1×10⁵个上述标记的AML原代细胞；每孔分别取100微升杀伤体系上清于酶标板中，使用多功能酶标仪(Varioscan Lux，ThermoFisher)检测荧光值(激发波长：495nm，发射波长：515nm)。

图5显示，与对照组CAR-T-1相比，具有不同抗原识别域的CAR-T-3对3个高(即AML1#)、中(即AML 2#)、低(即AML 3#)抗原密度的CD123阳性AML患者的原代细胞均具有显著增强的杀伤效率。说明具有CAR-T-3scFv的CD123 CAR-T细胞，能够有效提高对原代细胞的杀伤效率。

实施例6：改造后CD123 CAR-T在靶细胞刺激下的细胞因子表达

本实施例以检测具有不同抗原识别域的CD123 CAR-T在靶细胞AML原代细胞刺激下的细胞因子表达水平为例。

向48孔细胞培养板(Corning Incorporated，Corning，NY，USA)中，每孔加入1×10⁵个靶细胞AML原代细胞，此AML原代细胞的CD123表达率为99.87％、MFI为13298，并以E：T＝10：1的比例加入各组CAR-T细胞，置于37℃、5％CO₂培养箱中孵育5小时。取细胞上清，使用CBA试剂盒(BD Biosciences，San Jose，CA，USA)和流式细胞仪(NovoCyte 2060R，ACEABiosciences，San Diego，CA，USA)检测各种细胞因子的表达水平。

图6显示，与对照组CAR-T-1相比，具有不同抗原识别域的CAR-T-6组，在靶细胞刺激下，IL-2、TNF、IFN-γ的表达水平明显提高；其余各组CAR-T细胞的细胞因子表达，与对照组CAR-T-1相近。说明CAR-T-6的scFv设计有利于CD123 CAR-T细胞因子的表达。

以下表1和表2中为本发明中涉及的具体氨基酸序列。

表1示例性抗体的CDR和scFv序列

表2示例性嵌合抗原受体序列

参考文献:

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Ala Arg Gly Asn Trp Asp Asp Tyr

1 5

<210> 20

<211> 16

<212> PRT

<213> 合成序列

<400> 20

Ala Gly Gly Glu Gly Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr

1 5 10 15

<210> 21

<211> 17

<212> PRT

<213> 合成序列

<400> 21

Ala Arg Ser Pro Ser Tyr Tyr Gly Arg Ser Tyr Tyr Tyr Gly Met Asp

1 5 10 15

Tyr

<210> 22

<211> 6

<212> PRT

<213> 合成序列

<400> 22

Ala Arg Gly Met Asp Tyr

1 5

<210> 23

<211> 19

<212> PRT

<213> 合成序列

<400> 23

Ala Arg Glu Arg Gly Leu Tyr Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala

1 5 10 15

Met Asp Tyr

<210> 24

<211> 12

<212> PRT

<213> 合成序列

<400> 24

Ala Arg Gly Glu Gly Trp Leu Leu Pro Leu Ala Cys

1 5 10

<210> 25

<211> 11

<212> PRT

<213> 合成序列

<400> 25

Ala Ser Lys Ser Ile Ser Lys Asp Leu Ala Trp

1 5 10

<210> 26

<211> 11

<212> PRT

<213> 合成序列

<400> 26

Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala Trp

1 5 10

<210> 27

<211> 15

<212> PRT

<213> 合成序列

<400> 27

Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp

1 5 10 15

<210> 28

<211> 12

<212> PRT

<213> 合成序列

<400> 28

Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His Trp

1 5 10

<210> 29

<211> 11

<212> PRT

<213> 合成序列

<400> 29

Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp

1 5 10

<210> 30

<211> 17

<212> PRT

<213> 合成序列

<400> 30

Ser Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala

1 5 10 15

Trp

<210> 31

<211> 7

<212> PRT

<213> 合成序列

<400> 31

Gly Ser Thr Leu Gln Ser Gly

1 5

<210> 32

<211> 7

<212> PRT

<213> 合成序列

<400> 32

Thr Ser Thr Leu Gln Pro Gly

1 5

<210> 33

<211> 7

<212> PRT

<213> 合成序列

<400> 33

Ala Ser Asn Leu Glu Ser Gly

1 5

<210> 34

<211> 7

<212> PRT

<213> 合成序列

<400> 34

Thr Ser Asn Leu Ala Ser Gly

1 5

<210> 35

<211> 7

<212> PRT

<213> 合成序列

<400> 35

Ala Ser Gln Ser Ile Ser Glu

1 5

<210> 36

<211> 7

<212> PRT

<213> 合成序列

<400> 36

Ala Ser Thr Arg Asp Ser Gly

1 5

<210> 37

<211> 9

<212> PRT

<213> 合成序列

<400> 37

Gln His Asn Lys Tyr Pro Tyr Thr Phe

1 5

<210> 38

<211> 8

<212> PRT

<213> 合成序列

<400> 38

Gln Tyr Asp Asp Leu Tyr Thr Phe

1 5

<210> 39

<211> 9

<212> PRT

<213> 合成序列

<400> 39

Gln Ser Asn Glu Asp Pro Tyr Thr Phe

1 5

<210> 40

<211> 9

<212> PRT

<213> 合成序列

<400> 40

Gln Phe His Arg Ser Pro Arg Thr Phe

1 5

<210> 41

<211> 9

<212> PRT

<213> 合成序列

<400> 41

Asn Gly His Ser Phe Pro Tyr Thr Phe

1 5

<210> 42

<211> 9

<212> PRT

<213> 合成序列

<400> 42

Gln His Tyr Ser Thr Pro Trp Thr Phe

1 5

<210> 43

<211> 115

<212> PRT

<213> 合成序列

<400> 43

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

<210> 44

<211> 123

<212> PRT

<213> 合成序列

<400> 44

Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Thr Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Tyr Val His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Pro Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Phe Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gly Glu Gly Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 45

<211> 124

<212> PRT

<213> 合成序列

<400> 45

Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Met Thr Tyr

20 25 30

Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Phe

35 40 45

Gly Tyr Cys Asn Pro Tyr Asn Asp Gly Ile Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Ser Tyr Tyr Gly Arg Ser Tyr Tyr Tyr Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 46

<211> 113

<212> PRT

<213> 合成序列

<400> 46

Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Ser Gly Ser Ser Asn Ser Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

100 105 110

Ser

<210> 47

<211> 126

<212> PRT

<213> 合成序列

<400> 47

Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Ile Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Arg Gly Leu Tyr Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala

100 105 110

Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120 125

<210> 48

<211> 119

<212> PRT

<213> 合成序列

<400> 48

Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Ser Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Ser His Val Lys Ser Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asn Pro Tyr Asn Asn Ala Thr Ser Tyr Asn Gln Asn Phe

50 55 60

Lys Asp Lys Ala Ser Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Glu Gly Trp Leu Leu Pro Leu Ala Cys Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 49

<211> 107

<212> PRT

<213> 合成序列

<400> 49

Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly

1 5 10 15

Glu Thr Ile Thr Ile Asn 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 Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Met 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> 50

<211> 106

<212> PRT

<213> 合成序列

<400> 50

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gly Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Leu Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 51

<211> 111

<212> PRT

<213> 合成序列

<400> 51

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys

100 105 110

<210> 52

<211> 108

<212> PRT

<213> 合成序列

<400> 52

Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Val Phe Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Phe His Arg Ser Pro

85 90 95

Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 53

<211> 107

<212> PRT

<213> 合成序列

<400> 53

Asp Ile Gln Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Glu Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Phe Cys Gln Asn Gly His Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 54

<211> 113

<212> PRT

<213> 合成序列

<400> 54

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly

1 5 10 15

Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Asp Ser Gly Val

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln

85 90 95

His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 55

<211> 237

<212> PRT

<213> 合成序列

<400> 55

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser

130 135 140

Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser

145 150 155 160

Lys Asp Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu

165 170 175

Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe

180 185 190

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

195 200 205

Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Lys Tyr

210 215 220

Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230 235

<210> 56

<211> 244

<212> PRT

<213> 合成序列

<400> 56

Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Thr Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Tyr Val His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Pro Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Phe Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gly Glu Gly Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln

130 135 140

Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Gly Lys Val Thr Ile Thr

145 150 155 160

Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His

165 170 175

Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile His Tyr Thr Ser Thr Leu

180 185 190

Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gly Asp

195 200 205

Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr

210 215 220

Tyr Cys Leu Gln Tyr Asp Asp Leu Tyr Thr Phe Gly Gly Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys

<210> 57

<211> 250

<212> PRT

<213> 合成序列

<400> 57

Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Met Thr Tyr

20 25 30

Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Phe

35 40 45

Gly Tyr Cys Asn Pro Tyr Asn Asp Gly Ile Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Ser Tyr Tyr Gly Arg Ser Tyr Tyr Tyr Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr

130 135 140

Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile

145 150 155 160

Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met

165 170 175

Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Val Leu Ile Tyr

180 185 190

Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

195 200 205

Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu

210 215 220

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro Tyr Thr

225 230 235 240

Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys

245 250

<210> 58

<211> 236

<212> PRT

<213> 合成序列

<400> 58

Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Ser Gly Ser Ser Asn Ser Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Val Phe Leu Gly

130 135 140

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

145 150 155 160

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp

165 170 175

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

180 185 190

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

195 200 205

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Phe His Arg Ser Pro

210 215 220

Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230 235

<210> 59

<211> 248

<212> PRT

<213> 合成序列

<400> 59

Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Ile Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Arg Gly Leu Tyr Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala

100 105 110

Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly Asp Arg Val

145 150 155 160

Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp

165 170 175

Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys Tyr Ala

180 185 190

Ser Gln Ser Ile Ser Glu Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val

210 215 220

Gly Val Tyr Phe Cys Gln Asn Gly His Ser Phe Pro Tyr Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Leu Glu Ile Lys

245

<210> 60

<211> 247

<212> PRT

<213> 合成序列

<400> 60

Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Ser Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Ser His Val Lys Ser Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asn Pro Tyr Asn Asn Ala Thr Ser Tyr Asn Gln Asn Phe

50 55 60

Lys Asp Lys Ala Ser Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Glu Gly Trp Leu Leu Pro Leu Ala Cys Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser

130 135 140

Leu Ala Met Ser Val Gly Gln Lys Val Thr Met Ser Cys Lys Ser Ser

145 150 155 160

Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser

180 185 190

Thr Arg Asp Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly

195 200 205

Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala

210 215 220

Asp Tyr Phe Cys Gln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 61

<211> 244

<212> PRT

<213> 合成序列

<400> 61

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gly Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Leu Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser

115 120 125

Gly Pro Glu Leu Val Lys Thr Gly Ala Ser Val Lys Met Ser Cys Lys

130 135 140

Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Tyr Val His Trp Val Arg Gln

145 150 155 160

Ser His Gly Lys Ser Leu Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn

165 170 175

Gly Ala Thr Asn Tyr Asn Pro Lys Phe Lys Gly Lys Ala Thr Phe Thr

180 185 190

Val Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln Phe Lys Ser Leu Thr

195 200 205

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Gly Gly Glu Gly Tyr Tyr

210 215 220

Gly Tyr Asp Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val

225 230 235 240

Thr Val Ser Ser

<210> 62

<211> 42

<212> PRT

<213> 合成序列

<400> 62

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 63

<211> 112

<212> PRT

<213> 合成序列

<400> 63

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 64

<211> 501

<212> PRT

<213> 合成序列

<400> 64

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro

35 40 45

Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys

50 55 60

Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn Trp Val Lys Gln

65 70 75 80

Arg Pro Asp Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Tyr Asp

85 90 95

Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr

100 105 110

Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

115 120 125

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Asn Trp Asp Asp

130 135 140

Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly

145 150 155 160

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Ile Thr

165 170 175

Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly Glu Thr Ile Thr Ile

180 185 190

Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp Leu Ala Trp Tyr Gln

195 200 205

Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr

210 215 220

Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

225 230 235 240

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Met

245 250 255

Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr Thr Phe Gly Gly Gly

260 265 270

Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

275 280 285

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

290 295 300

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

305 310 315 320

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

325 330 335

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

340 345 350

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

355 360 365

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

370 375 380

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

385 390 395 400

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

405 410 415

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

420 425 430

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

435 440 445

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

450 455 460

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

465 470 475 480

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

485 490 495

Ala Leu Pro Pro Arg

500

<210> 65

<211> 508

<212> PRT

<213> 合成序列

<400> 65

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser

35 40 45

Gly Pro Glu Leu Val Lys Thr Gly Ala Ser Val Lys Met Ser Cys Lys

50 55 60

Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Tyr Val His Trp Val Arg Gln

65 70 75 80

Ser His Gly Lys Ser Leu Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn

85 90 95

Gly Ala Thr Asn Tyr Asn Pro Lys Phe Lys Gly Lys Ala Thr Phe Thr

100 105 110

Val Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln Phe Lys Ser Leu Thr

115 120 125

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Gly Gly Glu Gly Tyr Tyr

130 135 140

Gly Tyr Asp Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val

145 150 155 160

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

165 170 175

Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala

180 185 190

Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile

195 200 205

Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg

210 215 220

Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg

225 230 235 240

Phe Ser Gly Ser Gly Ser Gly Gly Asp Tyr Ser Phe Ser Ile Ser Asn

245 250 255

Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp

260 265 270

Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr

275 280 285

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

290 295 300

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

305 310 315 320

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

325 330 335

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

340 345 350

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

355 360 365

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

370 375 380

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

385 390 395 400

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu

405 410 415

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

420 425 430

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

435 440 445

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

450 455 460

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

465 470 475 480

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

485 490 495

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 66

<211> 514

<212> PRT

<213> 合成序列

<400> 66

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser

35 40 45

Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys

50 55 60

Ala Ser Gly Tyr Thr Phe Met Thr Tyr Val Ile His Trp Val Lys Gln

65 70 75 80

Lys Pro Gly Gln Gly Leu Glu Trp Phe Gly Tyr Cys Asn Pro Tyr Asn

85 90 95

Asp Gly Ile Asn Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr

100 105 110

Ser Asp Lys Ser Ser Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Thr

115 120 125

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Pro Ser Tyr Tyr

130 135 140

Gly Arg Ser Tyr Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

145 150 155 160

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

165 170 175

Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala

180 185 190

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser

195 200 205

Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro

210 215 220

Gly Gln Pro Pro Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser

225 230 235 240

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

245 250 255

Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys

260 265 270

Gln Gln Ser Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

275 280 285

Asp Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro

290 295 300

Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro

305 310 315 320

Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

325 330 335

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

340 345 350

Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu

355 360 365

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

370 375 380

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

385 390 395 400

Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys

405 410 415

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

420 425 430

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

435 440 445

Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

450 455 460

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

465 470 475 480

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

485 490 495

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

500 505 510

Pro Arg

<210> 67

<211> 500

<212> PRT

<213> 合成序列

<400> 67

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Lys Leu Gln Glu Ser

35 40 45

Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr

50 55 60

Val Thr Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn Trp Ile Arg

65 70 75 80

Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser

85 90 95

Gly Ser Ser Asn Ser Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr

100 105 110

Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr

115 120 125

Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Gly Met Asp Tyr Trp

130 135 140

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser

165 170 175

Pro Ala Ile Met Ser Val Phe Leu Gly Glu Arg Val Thr Met Thr Cys

180 185 190

Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln

195 200 205

Lys Pro Gly Ser Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu

210 215 220

Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser

225 230 235 240

Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr

245 250 255

Tyr Cys His Gln Phe His Arg Ser Pro Arg Thr Phe Gly Gly Gly Thr

260 265 270

Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

275 280 285

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

290 295 300

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

305 310 315 320

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

325 330 335

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys

340 345 350

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr

355 360 365

Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly

370 375 380

Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

385 390 395 400

Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

405 410 415

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

420 425 430

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

435 440 445

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

450 455 460

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

465 470 475 480

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

485 490 495

Leu Pro Pro Arg

500

<210> 68

<211> 512

<212> PRT

<213> 合成序列

<400> 68

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser

35 40 45

Gly Ala Glu Leu Val Arg Ser Gly Ala Ser Val Lys Met Ser Cys Lys

50 55 60

Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Asn Met His Trp Val Lys Gln

65 70 75 80

Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Gly Asn

85 90 95

Gly Gly Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr

100 105 110

Ala Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln Ile Ser Ser Leu Thr

115 120 125

Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Glu Arg Gly Leu Tyr

130 135 140

Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

145 150 155 160

Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

165 170 175

Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Thr

180 185 190

Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser

195 200 205

Gln Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu

210 215 220

Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Glu Ile

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser

245 250 255

Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Phe Cys Gln Asn

260 265 270

Gly His Ser Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

275 280 285

Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

290 295 300

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

305 310 315 320

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

325 330 335

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

340 345 350

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

355 360 365

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

370 375 380

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

385 390 395 400

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

405 410 415

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

420 425 430

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

435 440 445

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

450 455 460

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

465 470 475 480

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

485 490 495

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 69

<211> 511

<212> PRT

<213> 合成序列

<400> 69

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser

35 40 45

Gly Pro Glu Leu Val Lys Ser Gly Ala Ser Val Lys Ile Ser Cys Lys

50 55 60

Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Tyr Met His Trp Val Lys Gln

65 70 75 80

Ser His Val Lys Ser Leu Glu Trp Ile Gly Arg Ile Asn Pro Tyr Asn

85 90 95

Asn Ala Thr Ser Tyr Asn Gln Asn Phe Lys Asp Lys Ala Ser Leu Thr

100 105 110

Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu His Ser Leu Thr

115 120 125

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Glu Gly Trp Leu

130 135 140

Leu Pro Leu Ala Cys Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

165 170 175

Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly Gln

180 185 190

Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Ser

195 200 205

Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser

210 215 220

Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Asp Ser Gly Val Pro

225 230 235 240

Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

245 250 255

Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln His

260 265 270

Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

275 280 285

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

290 295 300

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

305 310 315 320

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

325 330 335

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

340 345 350

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

355 360 365

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

370 375 380

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

385 390 395 400

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln

405 410 415

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

420 425 430

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

435 440 445

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

450 455 460

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

465 470 475 480

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

485 490 495

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 70

<211> 508

<212> PRT

<213> 合成序列

<400> 70

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Ser His Pro Gln Phe Glu Lys Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser

35 40 45

Pro Ser Ser Leu Ser Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys

50 55 60

Lys Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys

65 70 75 80

Pro Gly Lys Gly Pro Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln

85 90 95

Pro Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gly Asp Tyr

100 105 110

Ser Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr

115 120 125

Cys Leu Gln Tyr Asp Asp Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu

130 135 140

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Thr

165 170 175

Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr

180 185 190

Asp Tyr Tyr Val His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu

195 200 205

Trp Ile Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Pro

210 215 220

Lys Phe Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser Thr

225 230 235 240

Ala Tyr Met Gln Phe Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr

245 250 255

Tyr Cys Ala Gly Gly Glu Gly Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met

260 265 270

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr

275 280 285

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

290 295 300

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

305 310 315 320

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

325 330 335

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

340 345 350

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

355 360 365

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

370 375 380

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

385 390 395 400

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu

405 410 415

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

420 425 430

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

435 440 445

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

450 455 460

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

465 470 475 480

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

485 490 495

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

Claims

1.一种靶向CD123的嵌合抗原受体，其氨基酸序列如SEQ ID NO:66所示。

2.一种核酸分子，其包含编码权利要求1所述的嵌合抗原受体的核苷酸序列。

3.一种载体，其包含权利要求2所述的核酸分子。

4.权利要求3所述的载体，其中，所述载体是病毒载体。

5.权利要求4所述的载体，其中，所述载体是慢病毒载体。

6.一种细胞，其经权利要求3所述的载体转染，从而包含权利要求2所述的核酸分子或权利要求3所述的载体。

7.权利要求6所述的细胞，其中，所述细胞为T细胞。

8.一种药物组合物，其包含权利要求1所述的嵌合抗原受体、权利要求2所述的核酸分子、权利要求3所述的载体或权利要求6所述的细胞，以及可药用载体。

9.权利要求1所述的嵌合抗原受体、权利要求2所述的核酸分子、权利要求3所述的载体或权利要求6所述的细胞用于制备治疗表达CD123的肿瘤的药物的用途。

10.权利要求9所述的用途，其中，所述表达CD123的肿瘤是急性髓系白血病。