CN107793480A - 一种抗cd19抗体及其制备方法和用途 - Google Patents
一种抗cd19抗体及其制备方法和用途 Download PDFInfo
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Abstract
本发明涉及生物技术领域，特别是涉及一种抗CD19抗体及其制备方法和用途。本发明提供一种抗CD19抗体，所述抗CD19抗体包括重链可变区和轻链可变区，重链可变区的互补决定区包括氨基酸序列如SEQ ID No.1所示的CDR‑H1、氨基酸序列如SEQ ID No.2或SEQ ID No.3所示的CDR‑H2和氨基酸序列如SEQ ID No.4所示的CDR‑H3，轻链可变区的互补决定区包括氨基酸序列如SEQ ID No.5所示的CDR‑L1、氨基酸序列如SEQ ID No.6所示的CDR‑L2和氨基酸序列如SEQ ID No.7所示的CDR‑L3。本发明发明人利用噬菌体展示技术对FMC63 scFv进行了亲和力成熟筛选，从而获得了高亲和力的对CD19的单链抗体。
Description
技术领域
本发明涉及生物技术领域，特别是涉及一种抗CD19抗体及其制备方法和用途。
背景技术
CD19抗原(B lymphocyte antigen CD19，CD19)是特异性表达于人类B细胞表面的一种蛋白。CD19广泛表达在各个发育时期的B细胞表面并起到了重要的作用:CD19作为B细胞受体(B cell receptor，BCR)共受体(co-receptor)，可以降低抗原介导的B细胞受体依赖性激活(antigen receptor-dependent stimulation)所需的信号阈值；B细胞受体激活依赖于CD19胞内区磷酸化继而招募Src激酶和PI3K激酶，从而彻底激活B细胞。完全成熟的B细胞称为浆细胞(plasma cell)，在完全成熟后浆细胞会丢失CD19抗原的表达。
B细胞恶性增殖相关的血液肿瘤包括：B细胞急性淋巴细胞白血病(acutelymphoblastic leukemia，B-ALL)、B细胞慢性淋巴细胞白血病(chronic lymphoblasticleukemia，B-CLL)以及B细胞淋巴瘤(B lymphoma，例如弥漫大B淋巴瘤及非霍奇金淋巴瘤)。B细胞急性淋巴细胞白血病和B细胞淋巴瘤是恶性程度很高的癌症，其特征为外周血、骨髓伴随高程度的B细胞恶性增殖以及全身性B细胞实体瘤的形成，严重干扰患者的血液循环系统。现今，对于B细胞肿瘤的治疗主要包括小分子靶向药物比如伊马替尼(费城染色体阳性)以及依鲁替尼(BTK inhibitor)、抗体药物比如利妥昔单抗(CD20antibody)以及骨髓移植(bone marrow transplantation)；在临床应用中，小分子药物以及抗体药物可以显著延长病人的生存期，为骨髓移植争取时间，但是部分病人会出现耐药性复发，至今临床上的药物对于耐药性复发的病人很难取得疗效。
嵌合抗原受体T细胞疗法(CART)属于新型的治疗方式，以病人T细胞为基础在体外扩增并进行基因工程改造，CART细胞能够识别肿瘤细胞特异性抗原(例如CD19)，激活后可以特异性裂解肿瘤细胞达到杀伤肿瘤目的，其特点为治疗周期短、疗效较快、完全响应率高、能够为病人争取足够的时间等待骨髓移植，甚至彻底治愈。嵌合抗原受体组成为单链抗体(scFv)-铰链区(hinge region)-跨膜区(transmembrane domain)-共刺激结构域(co-stimulatory domain)-必要刺激结构域(essential signaling domain)，其中对抗原具有高亲单链抗体为嵌合抗原受体(CAR)重要组成部分，scFv对于抗原的亲和力决定了CART细胞能否激活以及杀伤肿瘤细胞，因此对于抗原特异性scFv进行亲和力成熟的筛选尤其重要。
FMC63-mIgG2a是上个世纪通过动物免疫获得抗CD19的鼠源抗体。FMC63 scFv已经成功被应用于抗CD19-CAR构造，例如CTL019(Norvatis)、JCAR014-017(JunoTherapeutics)及KTE-C19(KITE Pharma)。CTL019、JCAR015及KTE-C19在B细胞恶性肿瘤(ALL、DLBCL、NHL等)的临床试验取得了较好的结果，完全缓解(complete remission)病人比例70-94％(1-3个月)。病人在接受表达鼠源嵌合抗原受体T细胞输注后，一般可在1-3个月内会获得完全缓解，12个月仍然保持缓解一般只能达到30％，复发的原因主要为：1.肿瘤细胞表面CD19抗原发生剪切(集中发生在exon2及exon4)或丢失，使得体内的CART细胞不能识别肿瘤细胞(FMC63识别epitope丢失)。2.肿瘤细胞表面仍然表达CD19，CART细胞在体内持续性较差，无法在患者体内持续扩增，可能原因自CAR鼠源框架引发体液免疫生成HAMA抗体(Human anti mouse antibody，HAMA)造成的CART细胞活性阻断或清除；病人在获得完全缓解之前，鼠源框架引起体内正常B细胞免疫反应并发育为浆细胞(Plasma Cell)，分泌大量HAMA抗体；由于浆细胞不表达CD19抗原，因此CART细胞无法清除浆细胞，从而导致了抗体介导的CART细胞活性封闭(Blocking)及清除(Elimination)。
对鼠源抗体进行人源化改造可以有效降低免疫源性，可能提高CART细胞在体内的持续性。专利号为US20140271635中，对FMC63进行了人源化设计(人源Germline框架下CDR移植)，其重链可变区为VH4 4-59(Vbase2)，轻链可变区为VK3_L25(Vbase2)，通过体外结合测试共得到了12种克隆(C2136-C2147)与CD19保持结合活性，其中C2146克隆为优选克隆，其CDR区域中HCDR2发生了单氨基酸突变(YNSAL→YNSSL)；经过scFv细胞结合实验表明C2146克隆与FMC63克隆相比，其亲和力下降2-3倍。
目前，人源化抗CD19嵌合抗原受体T细胞(简称huCART19)用于治疗复发难治CD19阳性白血病或淋巴瘤临床试验正在开展(NCT02374333，宾夕法尼亚大学)；2016年第58届ASH大会中Shannon L Maude等人发布了huCART19的临床结果，在复发难治的接受过鼠源CD19CART治疗没有响应的患者人群中，重新给予输注huCART19，可以获得64％完全缓解(7/11)。此次临床结果提示人源化huCART19可能保持了较低的免疫源性，避免了HAMA抗体介导CART细胞在患者体内的阻断或清除效应，从而在既往接受鼠源CD19-CART没有响应的患者中产生较高的完全缓解率。在人源化过程中，对鼠源框架进行了替换，huCART19对CD19抗原的亲和力有所降低，因此在保证较低的免疫源性基础上对huCART19scFv亲和力提高可能提高其药效及反应率。
已有文献报导提高scFv对抗原的亲和力可以提高CART细胞对于肿瘤细胞的识别、特异性激活、细胞因子分泌以及杀伤效率(Hudecek等，2013；Lynn RC等，2016)。本发明利用噬菌体展示技术对FMC63 scFv及C2146 scFv进行了亲和力成熟筛选，筛选的scFv可以提高对CD19的亲和力；基于筛选的scFv改造成的CD19-CART可以提高对CD19阳性的肿瘤细胞的杀伤效率、扩增效率及持续性。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种抗CD19抗体及其制备方法和用途，用于解决现有技术中的问题。
为实现上述目的及其他相关目的，本发明一方面提供一种抗CD19抗体，所述抗CD19抗体包括重链可变区和轻链可变区，所述抗CD19抗体具有如下技术特征中的一个或多个：
<1>重链可变区的互补决定区包括氨基酸序列如SEQ ID No.1所示的CDR-H1；
<2>重链可变区的互补决定区包括氨基酸序列如SEQ ID No.2或SEQ ID No.3所示的CDR-H2；
<3>重链可变区的互补决定区包括氨基酸序列如SEQ ID No.4所示的CDR-H3；
<4>轻链可变区的互补决定区包括氨基酸序列如SEQ ID No.5所示的CDR-L1；
<5>轻链可变区的互补决定区包括氨基酸序列如SEQ ID No.6所示的CDR-L2；
<6>轻链可变区的互补决定区包括氨基酸序列如SEQ ID No.7所示的CDR-L3。
GFSLEDYGVS(SEQ ID No.1)
VIWGSETTYYNSALKS(SEQ ID No.2)
VIWGSETTYYNSSLKS(SEQ ID No.3)
HYYYGGSYAMDY(SEQ ID No.4)
RASQDISKYLN(SEQ ID No.5)
HTSRLHS(SEQ ID No.6)
QQGKTFPLT(SEQ ID No.7)
上述SEQ ID No.1-7所给出的序列中，突变以下划线标出。
CDR(互补决定区，complementarity determining region)通常指抗体中在空间结构上可以与抗原决定簇形成互补的区域。抗体中的可变性通常并不均匀地分布在整个抗体的可变区中，单克隆抗体的重链可变区和轻链可变区通常均具有3个超变区(hypervariable region，HVR)，这些区域在空间结构上通常可以与抗原决定簇形成互补，所以超变区也被称为互补决定区(complementarity determining region，CDR)，即重链可变区通常包括三个互补决定区，即HCDR1、HCDR2和HCDR3，轻链可变区通常包括三个互补决定区，即LCDR1、LCDR2和LCDR3。
在本发明某些实施方式中，所述抗CD19抗体的重链可变区的互补决定区包括氨基酸序列如SEQ ID No.1所示的CDR-H1、氨基酸序列如SEQ ID No.2或SEQ ID No.3所示的CDR-H2和氨基酸序列如SEQ ID No.4所示的CDR-H3。
在本发明某些实施方式中，所述抗CD19抗体的轻链可变区的互补决定区包括氨基酸序列如SEQ ID No.5所示的CDR-L1、氨基酸序列如SEQ ID No.6所示的CDR-L2和氨基酸序列如SEQ ID No.7所示的CDR-L3。
在本发明某些实施方式中，重链可变区的互补决定区包括氨基酸序列如SEQ IDNo.1所示的CDR-H1、氨基酸序列如SEQ ID No.2或SEQ ID No.3所示的CDR-H2和氨基酸序列如SEQ ID No.4所示的CDR-H3，轻链可变区的互补决定区包括氨基酸序列如SEQ ID No.5所示的CDR-L1、氨基酸序列如SEQ ID No.6所示的CDR-L2和氨基酸序列如SEQ ID No.7所示的CDR-L3。
在本发明某些实施方式中，所述抗CD19抗体为单克隆抗体。单克隆抗体通常指一个抗体的群体，该群体中所包括的抗体是基本相同的(除少数可能存在的天然发生的突变外)。单克隆抗体通常针对抗原上特定的决定簇。
在本发明某些实施方式中，所述抗CD19抗体为单链抗体(single chain Fv，scFv)。单链抗体通常可以为包含抗体的VH(重链可变区)和VL(轻链可变区)的多肽链。通常来说，单链抗体还可以包括连接肽(linker)，连接肽通常位于VH和VL之间，以使scFv形成能与抗原结合的期望结构。例如，所述抗CD19抗体可以包括VH和VL，VH和VL之间可以设有连接肽，所述单链抗CD19抗体自N段至C端可以依次包括VH、连接肽和VL，所述抗CD19单链抗体自N段至C端也可以依次包括VL、连接肽和VH。所述连接肽可以是本领域中各种适用于形成scFv的连接肽，例如，所述连接肽可以是G4S3linker，所述G4S3linker的选择或设计可以参考文献Michel Sadelain etc，Science Translational Medicine，2013；Carl H.June etc，Science Translational Medicine，2015。
在本发明某些实施方式中，所述抗CD19抗体是鼠源的，更具体的，所述抗CD19抗体衍生自CD19特异性的单克隆抗体FMC63(VH：Y14283.1，VL:Y14284.1)，其核苷酸序列如SEQID No.8所示，氨基酸序列如SEQ ID No.9所示。
GAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACA(SEQ ID No.8)
(SEQ ID No.9，其中粗体加下划线部分为连接肽，连接肽之前为重链可变区，连接肽之后为轻链可变区，下划线部分依次为CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2、CDR-L3)
在本发明某些实施方式中，所述抗CD19抗体是人源的，更具体的，所述抗CD19抗体衍生自C2146序列(VH及VL序列参见US20140271635，其VL-VH序列参照第71条序列，氨基酸序列参照第83条序列)，其核苷酸序列如SEQ ID No.10所示，氨基酸序列如SEQ ID No.11所示。
C2146单链抗体(VL-VH)核苷酸序列为：
GAAATTGTGATGACCCAGTCACCCGCCACTCTTAGCCTTTCACCCGGTGAGCGCGCAACCCTGTCTTGCAGAGCCTCCCAAGACATCTCAAAATACCTTAATTGGTATCAACAGAAGCCCGGACAGGCTCCTCGCCTTCTGATCTACCACACCAGCCGGCTCCATTCTGGAATCCCTGCCAGGTTCAGCGGTAGCGGATCTGGGACCGACTACACCCTCACTATCAGCTCACTGCAGCCAGAGGACTTCGCTGTCTATTTCTGTCAGCAAGGGAACACCCTGCCCTACACCTTTGGACAGGGCACCAAGCTCGAGATTAAAGGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGCCAGGTCCAACTCCAAGAAAGCGGACCGGGTCTTGTGAAGCCATCAGAAACTCTTTCACTGACTTGTACTGTGAGCGGAGTGTCTCTCCCCGATTACGGGGTGTCTTGGATCAGACAGCCACCGGGGAAGGGTCTGGAATGGATTGGAGTGATTTGGGGCTCTGAGACTACTTACTACAATTCATCCCTCAAGTCACGCGTCACCATCTCAAAGGACAACTCTAAGAATCAGGTGTCACTGAAACTGTCATCTGTGACCGCAGCCGACACCGCCGTGTACTATTGCGCTAAGCATTACTATTATGGCGGGAGCTACGCAATGGATTACTGGGGACAGGGTACTCTGGTCACCGTGTCCAGC(SEQ ID No.10)
氨基酸序列为：
(SEQ ID No.11，其中粗体加下划线部分为连接肽，连接肽之前为轻链可变区，连接肽之后为重链可变区，下划线部分依次为CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2、CDR-H3)
在本发明某些实施方式中，重链可变区和轻链可变区中还可以包括框架区，所述框架区可以位于互补决定区之间，也可以位于互补决定区的两端。在本发明一具体实施方式中，所述框架区的序列与FMC63或C2146序列的框架区序列一致，或为FMC63或C2146的框架区序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、或1-3个)氨基酸而得到的框架区序列，该框架区序列与FMC63的框架区序列或C2146的框架区序列可以具有80％、85％、90％、93％、95％、97％、或99％以上的同源性。
在本发明某些实施方式中，所述抗CD19抗体的重链可变区的氨基酸序列包括：
a)如SEQ ID No.64-69其中之一所示的氨基酸序列；或
b)与SEQ ID No.64-69其中之一所示的氨基酸序列具有80％以上同源性、且具有a)所限定的氨基酸序列功能的氨基酸序列。
具体的，所述b)中的氨基酸序列具体指：如SEQ ID No.64-69其中之一所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、或1-3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、或1-3个)氨基酸而得到的，且具有如SEQ ID No.64-69其中之一所示的氨基酸序列功能的氨基酸序列。所述b)中的氨基酸序列可与SEQ IDNo.64-69其中之一具有80％、85％、90％、93％、95％、97％、或99％以上的同源性。
在本发明某些实施方式中，所述抗CD19抗体的轻链可变区的氨基酸序列包括：
c)如SEQ ID No.70-75其中之一所示的氨基酸序列；或
d)与SEQ ID No.70-75其中之一所示的氨基酸序列具有80％以上同源性、且具有c)所限定的氨基酸序列功能的氨基酸序列。
具体的，所述d)中的氨基酸序列具体指：如SEQ ID No.70-75其中之一所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、或1-3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、或1-3个)氨基酸而得到的，且具有如SEQ ID No.70-75其中之一所示的氨基酸序列功能的氨基酸序列。所述b)中的氨基酸序列可与SEQ IDNo.70-75其中之一具有80％、85％、90％、93％、95％、97％、或99％以上的同源性。
本发明另一方面提供一种分离的多核苷酸，编码所述抗CD19抗体的重链可变区和/或轻链可变区或全长氨基酸。
本发明另一方面提供一种构建体，含有所述分离的多核苷酸。
在本发明某些实施方式中，所述构建体由所述分离的多核苷酸插入到表达载体的多克隆位点构建而成。本发明中的表达载体通常指本领域熟知的各种市售表达载体，例如可以是细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。
在本发明某些实施方式中，所述表达载体选自GV401表达载体(GV401为市售载体，供应商为吉凯基因)。
本发明另一方面提供一种抗体的表达系统，所述表达系统含有所述构建体或基因组中整合有外源的所述多核苷酸。任何适用于表达载体进行表达的细胞都可以作为宿主细胞，例如，所述宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。
在本发明某些实施方式中，宿主细胞选自T细胞、NK细胞中的一种或多种的组合。
本发明另一方面提供所述抗CD19抗体的制备方法，包括如下步骤：在适合表达所述抗体的条件下，培养所述的抗体的表达系统，从而表达出所述的抗体，纯化分离出所述的抗体。
本发明中所用的宿主细胞均为现有技术，可通过商业途径直接获取，培养中所用的培养基亦为各种常规培养基，本领域技术人员可根据经验选择适用的培养基，在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明另一方面提供所述抗CD19抗体在制备或筛选治疗药物中的用途、或制备诊断药物中的用途。
所述治疗药物可以是以CD19抗原为作用靶标，结合或作用于所述CD19抗原，从而治疗和/或预防适应症的药物。
在本发明某些实施方式中，所述治疗药物可以是肿瘤治疗药物。所述肿瘤治疗药物可以是以肿瘤细胞表面功能性表达的CD19抗原为靶标，结合或作用于CD19抗原，从而治疗和/或预防肿瘤的药物。所述肿瘤可以是急性淋巴型白血病、慢性淋巴型白血病、B淋巴瘤、或者其他与B细胞恶性增殖相关的肿瘤。
在本发明某些实施方式中，所述治疗药物为嵌合抗原受体(CAR，chimericantigen receptor)细胞治疗药物。
所述嵌合抗原受体细胞治疗药物通常包括嵌合抗原受体细胞，所述嵌合抗原受体细胞可以是嵌合抗原受体T细胞、嵌合抗原受体NK细胞等，所述嵌合抗原受体T细胞通常包括T淋巴细胞，其还包括嵌合抗原受体。所述嵌合抗原受体NK细胞通常包括NK细胞，其还包括嵌合抗原受体。所述嵌合抗原受体包括跨膜域、胞内域和胞外域。在本发明某些实施方式中，所述胞外域包括所述抗CD19抗体，即所述嵌合抗原受体细胞可以在细胞表面表达所述抗CD19抗体，从而可以引导该细胞对表达CD19抗原的细胞(例如，肿瘤细胞)进行作用的药物。所述对表达CD19抗原的细胞进行作用可以是杀死表达CD19抗原的细胞等。
所以诊断药物具体指针对作用靶标CD19抗原，以CD19抗原作为生物标志物进行诊断的试剂。
本发明另一方面提供一种分离的多肽，所述多肽包括跨膜域、胞内域和胞外域，所述胞外域包括所述抗CD19抗体。
在本发明某些实施方式中，所述多肽为嵌合抗原受体。
在本发明某些实施方式中，所述跨膜域可以包括CD8α(NM_001145873)、CD28(NM_006139)、DAP10(NM_014266)等蛋白分子的跨膜结构域。
在本发明某些实施方式中，所述胞内域可以包括共刺激结构域和/或信号结构域，例如，所述胞内域可以包括4-1BB(NM_001561)、CD28(NM_006139)、OX40(NM_003327)、ICOS(NM_012092)、CD3zeta(NM_198053)、DAP10(NM_014266)等蛋白分子的信号转导结构域。
在本发明某些实施方式中，所述多肽自N端至C端依次包括所述抗CD19单链抗体、跨膜域、胞内域。在本发明一些具体实施方式中，所述多肽自N端至C端依次包括所述抗CD19单链抗体、CD8α跨膜区、4-1BB共刺激结构域、CD3zeta信号结构域。在本发明一具体实施方式中，所述多肽自N端至C端依次包括所述抗CD19单链抗体、CD28跨膜区、CD28共刺激结构域、CD3zeta信号结构域。在本发明另一具体实施方式中，所述多肽自N端至C端依次包括所述抗CD19单链抗体、CD8α跨膜区、OX40共刺激结构域、CD3zeta信号结构域。在本发明另一具体实施方式中，所述多肽自N端至C端依次包括所述抗CD19单链抗体、CD8α跨膜区、ICOS共刺激结构域、CD3zeta信号结构域。在本发明另一具体实施方式中，所述多肽自N端至C端依次包括所述抗CD19单链抗体、CD8α跨膜区、4-1BB共刺激结构域、CD28共刺激结构域、CD3zeta。在本发明另一具体实施方式中，所述多肽自N端至C端依次包括所述抗CD19单链抗体、CD28跨膜区、CD28共刺激结构域、OX40共刺激结构域、CD3zeta信号结构域。
本发明另一方面一种T淋巴细胞，所述T淋巴细胞含膜结合的所述多肽。
在本发明某些实施方式中，所述多肽为嵌合抗原受体。
所述T淋巴细胞通常可以表达所述多肽，其通常可以结合于CD19抗原，更具体可以通过包含所述抗CD19抗体的胞外域结合于CD19抗原，当所述多肽结合于所述CD19抗原时，所述T淋巴细胞通常可以活化和/或刺激从而得以增殖。在本发明某些实施方式中，所述胞外域包括所述抗CD19抗体，即所述嵌合抗原受体T细胞可以在T淋巴细胞表面表达所述抗CD19抗体，从而可以引导T淋巴细胞对表达CD19抗原的细胞(例如，肿瘤细胞)进行作用的，所述作用可以是杀死表达CD19抗原的细胞等。
本发明另一方面一种NK细胞，所述NK细胞含膜结合的所述多肽。
在本发明某些实施方式中，所述多肽为嵌合抗原受体。
所述NK细胞通常可以表达所述多肽，其通常可以结合于CD19抗原，更具体可以通过包含所述抗CD19抗体的胞外域结合于CD19抗原，当所述多肽结合于所述抗原时，所述NK细胞通常可以活化和/或刺激从而得以增殖。在本发明某些实施方式中，所述胞外域包括所述抗CD19抗体，即所述嵌合抗原受体NK细胞可以在NK细胞表面表达所述抗CD19抗体，从而可以引导NK细胞对表达CD19抗原的细胞(例如，肿瘤细胞)进行作用的，所述作用可以是杀死表达CD19抗原的细胞等。
本发明另一方面提供一种诊断试剂盒，包含诊断有效剂量的所述抗CD19抗体或其免疫偶联物。有效量通常指能够提供诊断效益的量。
所以诊断试剂盒通常可以针对作用靶标CD19抗原，以CD19抗原作为生物标志物进行诊断。所述诊断试剂盒还可以包括抗CD19抗体的标记物，所述抗CD19抗体的标记物通常可以用于标记抗CD19抗体，可选用的标记物的种类包括但不限于荧光标记物、放射性标记物、酶标标记物、化学发光性标记物等中的一种或多种的组合。根据试剂盒的检测原理，所述试剂盒通常还可包含检测所需的一种或多种试剂。此外，所述试剂盒中还可根据需要包括：容器、对照物(阴性或阳性对照)、缓冲剂、助剂等，本领域技术人员可根据具体情况对其进行选择。
本发明发明人利用噬菌体展示技术对FMC63和C2146 scFv进行了亲和力成熟筛选，从而获得了高亲和力的对CD19的单链抗体，且亲和力成熟的单链抗体仍然结合FMC63或C2146识别的抗原决定簇，从而可以提示这些单链抗体的抗原结合位点与FMC63和C2146一致。此外，本发明发明人进一步将高亲和力的单链抗体改造成嵌合抗原受体，例如利用表达抗CD19嵌合抗原受体的T细胞、NK细胞用于表达CD19的血液癌症(B-ALL，B-CLL，B-Lymphoma等)治疗，从而验证了突变后的嵌合抗原受体能够提高对肿瘤细胞的杀伤能力。
附图说明
图1显示为实施例1中pCANTAB5E载体示意图(注：pCANTAB5E载体Etag末端与Amber终止密码子之间，通过插入Myc肽段(GAGCAGAAGCTGATCTCAGAGGAGGACCTG)用于scFv在培养基上清中的检测)。
图2显示为实施例3中FMC63突变库噬菌体第四轮淘选克隆数量分析结果示意图，CloneCount代表挑取克隆中唯一序列的数量，从左至右的深色柱分别是4B10及8A10突变。
图3显示为实施例3中C2146突变库噬菌体第四轮淘选克隆数量分析结果示意图，从左至右的深色柱分别是7F12、7E11、7C3。
图4、图5显示为噬菌体结合ELISA实验结果示意图。
图6显示为实施例5噬菌体竞争ELISA筛选结果示意图。
图7显示为实施例7中FMC63，4B10，8A10及C21hIgG1与Raji细胞结合结果示意图。
图8显示为实施例7中C2146，7F12，7C3及7E11hIgG1与K562-CD19细胞结合结果示意图。
图9显示为实施例7中FMC63及C21hIgG1与Raji细胞结合结果示意图。
图10显示为实施例7中C2146及7E11hIgG1与Raji细胞结合结果示意图。
图11显示为GV401载体结构示意图。
图12-13显示为实施例9肿瘤细胞裂解效率示意图。
图14显示为实施例10中Raji荷瘤晚期模型药效结果示意图。
图15显示为实施例10中小鼠外周血IFNγ释放检测结果示意图。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring HarborLaboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS INMOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；theseries METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATINSTRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS INENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，AcademicPress，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，ChromatinProtocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1
FMC63 scFv和C2146 scFv的重链(H)及轻链(L)的CDR1、CDR2、CDR3突变库的构建：
FMC63及C2146重链及轻链CDR区域的选择基于可变区的氨基酸计数法(模板为单克隆抗体FMC63，VH：Y14283.1，VL:Y14284.1；C2146单链抗体序列选自US20140271635)，Kabat计数(Kabat number scheme.Bioinf.org.uk)列表如下：
表1
以pCAN-FMC63 scFv质粒和pCAN-C2146 scFv(由模板序列插入pCANTAB 5E质粒(购自GE)的多克隆位点构建，FMC63模板序列为：VH：Y14283.1，VL:Y14284.1，C2146模版序列参见US20140271635)为模板，利用随机引物PCR引入突变，FMC63突变库引物和C2146突变库引物分别如表2和表3所示。获得的FMC63 scFv的重链(H)及轻链(L)的CDR1、CDR2、CDR3突变库PCR产物分别命名为H1、H2、H3、L1、L2和L3。获得的C2146 scFv的重链(VH)及轻链(VL)的CDR1、CDR2、CDR3突变库PCR产物分别命名为huH1、huH2、huH3、huL1、huL2和huL3。用Sfi I和Not I对PCR产物及pCANTAB 5E酶切回收后，经T4DNA连接酶16℃连接过夜。连接产物电转至TG1感受态细胞，2xYT培养基重悬并于37℃复苏1h后，取菌液梯度稀释(1/100、1/1000、1/10000)进行平板计数，得到各个突变库库容至少108-9，其余菌液全部涂布2xYT(GA)平板(葡萄糖2％，青霉素100ug/ml)。从上述突变库中分别随机挑取20个单克隆送测序，通过序列分析克隆多样性。
表2
表3
注：C2146库构建时，将huH1，huH2，huH3及huL1，huL2，huL3PCR产物进行混合，产生可能同时在HCDR及LCDR同时突变的克隆。
实施例2
噬菌体抗体库的淘选：
加入20nM CD19-his-biotin抗原与FMC63 scFv或C2146 scFv噬菌体抗体库室温孵育2h，再将混合物转入链霉亲和素磁珠(Dynabeads M-280Streptavidin)中室温共孵育15min。PBST-PBS洗去未结合的噬菌体，再加入胰酶37℃作用30min，从而洗脱下结合的噬菌体。将胰酶消化洗脱下的噬菌体感染4ml对数期的TG1菌体，37℃静置30min，取部分菌液梯度稀释(1/10，1/100，1/1000)进行平板计数，其余菌液全部涂布2xYT(GA)平板，用于下一轮的包装。包装后的噬菌体可用于下一轮的淘选，共进行4轮淘选富集，每轮淘选10倍稀释梯度降低抗原浓度，并逐轮增加PBST-PBS洗涤次数(四轮淘选的CD19-his-biotin抗原浓度分别为20nM、2nM、0.2nM和0.02nM，PBST-PBS洗涤次数分别为7，10，15，20次)。
实施例3
高亲和力scFv的筛选和鉴定：
经过四轮的淘选后，随机挑取单克隆，IPTG诱导后取上清进行ELISA筛选：将1ug/mlAvidin包被96孔板过夜，用PBST洗2次，将100ul 1ug/ml CD19-biotin加入Avidin包被的96孔板，孵育1小时后，用PBST洗2次后，加入IPTG诱导后上清孵育1小时，PBST洗4次，加入1:5000稀释抗Myc抗体100ul孵育30分钟，PBST洗4次后，加入TMB底物反应10分钟，加入1M硫酸溶液终止反应，读取OD450nm光吸收。挑取阳性信号(OD450nm光吸收)至少2倍大于阴性信号的克隆送测序，分析测序结果，提取出富集较多的CDR区域对应的克隆。克隆数量与CDR突变关系如图2和图3所示。
从以上序列富集分析，FMC63突变库中4B10(HCDR1)及8A10(LCDR3)序列与C2146突变库中7F12(HCDR1)及7C3(LCDR3)序列完全一致，上述序列的信息具体如下。在C2146突变库筛选结果分析发现(见表3的标注信息)，HCDR1及LCDR3同时突变克隆7E11富集明显，如图3所示。以上结果，提示FMC63及C2146突变库中HCDR1(4B10，7F12)、LCDR3(8A10，7C3)及两者组合突变(7E11)，有利于对CD19的结合。
克隆号：4B10
GAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGTTTTCATTAGAGGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACA(SEQ ID No.52)
(SEQ ID No.58，其中粗体加下划线部分为连接肽，连接肽之前为重链可变区，连接肽之后为轻链可变区，下划线部分依次为CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2、CDR-L3)EVKLQESGPGLVAPSQSLSVTCTVSGFSLEDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID No.64)
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT(SEQ ID No.70)
克隆号：8A10
GAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCCGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAAGACGTTTCCGCTTACGTTCGGAGGGGGGACCAAGCTGGAGATCACA(SEQ ID No.53)
(SEQ ID No.59，其中粗体加下划线部分为连接肽，连接肽之前为重链可变区，连接肽之后为轻链可变区，下划线部分依次为CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2、CDR-L3)
EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGPEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID No.65)
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTFPLTFGGGTKLEIT(SEQ ID No.71)
克隆号：C21(4B10&8A10组合突变)
GAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGTTTTCATTAGAGGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAAGACGTTTCCGCTTACGTTCGGAGGGGGGACCAAGCTGGAGATCACA(SEQ ID No.54)
(SEQ ID No.60，其中粗体加下划线部分为连接肽，连接肽之前为重链可变区，连接肽之后为轻链可变区，下划线部分依次为CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2、CDR-L3)
EVKLQESGPGLVAPSQSLSVTCTVSGFSLEDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID No.66)
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTFPLTFGGGTKLEIT(SEQ ID No.72)
克隆号7F12
GAAATTGTGATGACCCAGTCACCCGCCACTCTTAGCCTTTCACCCGGTGAGCGCGCAACCCTGTCTTGCAGAGCCTCCCAAGACATCTCAAAATACCTTAATTGGTATCAACAGAAGCCCGGACAGGCTCCTCGCCTTCTGATCTACCACACCAGCCGGCTCCATTCTGGAATCCCTGCCAGGTTCAGCGGTAGCGGATCTGGGACCGACTACACCCTCACTATCAGCTCACTGCAGCCAGAGGACTTCGCTGTCTATTTCTGTCAGCAAGGGAACACCCTGCCCTACACCTTTGGACAGGGCACCAAGCTCGAGATTAAAGGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGCCAGGTCCAACTCCAAGAAAGCGGACCGGGTCTTGTGAAGCCATCAGAAACTCTTTCACTGACTTGTACTGTGAGCGGATTTTCTCTCGAGGATTACGGGGTGTCTTGGATCAGACAGCCACCGGGGAAGGGTCTGGAATGGATTGGAGTGATTTGGGGCTCTGAGACTACTTACTACAATTCATCCCTCAAGTCACGCGTCACCATCTCAAAGGACAACTCTAAGAATCAGGTGTCACTGAAACTGTCATCTGTGACCGCAGCCGACACCGCCGTGTACTATTGCGCTAAGCATTACTATTATGGCGGGAGCTACGCAATGGATTACTGGGGACAGGGTACTCTGGTCACCGTGTCCAGC(SEQ ID No.55)
(SEQ ID No.61，其中粗体加下划线部分为连接肽，连接肽之前为轻链可变区，连接肽之后为重链可变区，下划线部分依次为CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2、CDR-H3)
EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK(SEQ ID No.73)
QVQLQESGPGLVKPSETLSLTCTVSGFSLEDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(SEQ ID No.67)
克隆号7C3
GAAATTGTGATGACCCAGTCACCCGCCACTCTTAGCCTTTCACCCGGTGAGCGCGCAACCCTGTCTTGCAGAGCCTCCCAAGACATCTCAAAATACCTTAATTGGTATCAACAGAAGCCCGGACAGGCTCCTCGCCTTCTGATCTACCACACCAGCCGGCTCCATTCTGGAATCCCTGCCAGGTTCAGCGGTAGCGGATCTGGGACCGACTACACCCTCACTATCAGCTCACTGCAGCCAGAGGACTTCGCTGTCTATTTCTGTCAGCAAGGGAAGACCTTTCCCCTTACCTTTGGACAGGGCACCAAGCTCGAGATTAAAGGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGCCAGGTCCAACTCCAAGAAAGCGGACCGGGTCTTGTGAAGCCATCAGAAACTCTTTCACTGACTTGTACTGTGAGCGGAGTGTCTCTCCCCGATTACGGGGTGTCTTGGATCAGACAGCCACCGGGGAAGGGTCTGGAATGGATTGGAGTGATTTGGGGCTCTGAGACTACTTACTACAATTCATCCCTCAAGTCACGCGTCACCATCTCAAAGGACAACTCTAAGAATCAGGTGTCACTGAAACTGTCATCTGTGACCGCAGCCGACACCGCCGTGTACTATTGCGCTAAGCATTACTATTATGGCGGGAGCTACGCAATGGATTACTGGGGACAGGGTACTCTGGTCACCGTGTCCAGC(SEQ ID No.56)
(SEQID No.62，其中粗体加下划线部分为连接肽，连接肽之前为轻链可变区，连接肽之后为重链可变区，下划线部分依次为CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2、CDR-H3)
EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGKTFPLTFGQGTKLEIK(SEQ ID No.74)
QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(SEQ ID No.68)
克隆号7E11(7F12&7C3组合突变)
GAAATTGTGATGACCCAGTCACCCGCCACTCTTAGCCTTTCACCCGGTGAGCGCGCAACCCTGTCTTGCAGAGCCTCCCAAGACATCTCAAAATACCTTAATTGGTATCAACAGAAGCCCGGACAGGCTCCTCGCCTTCTGATCTACCACACCAGCCGGCTCCATTCTGGAATCCCTGCCAGGTTCAGCGGTAGCGGATCTGGGACCGACTACACCCTCACTATCAGCTCACTGCAGCCAGAGGACTTCGCTGTCTATTTCTGTCAGCAAGGGAAGACCTTTCCCCTTACCTTTGGACAGGGCACCAAGCTCGAGATTAAAGGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGCCAGGTCCAACTCCAAGAAAGCGGACCGGGTCTTGTGAAGCCATCAGAAACTCTTTCACTGACTTGTACTGTGAGCGGATTTTCTCTCGAGGATTACGGGGTGTCTTGGATCAGACAGCCACCGGGGAAGGGTCTGGAATGGATTGGAGTGATTTGGGGCTCTGAGACTACTTACTACAATTCATCCCTCAAGTCACGCGTCACCATCTCAAAGGACAACTCTAAGAATCAGGTGTCACTGAAACTGTCATCTGTGACCGCAGCCGACACCGCCGTGTACTATTGCGCTAAGCATTACTATTATGGCGGGAGCTACGCAATGGATTACTGGGGACAGGGTACTCTGGTCACCGTGTCCAGC(SEQ ID No.57)
(SEQID No.63，其中粗体加下划线部分为连接肽，连接肽之前为轻链可变区，连接肽之后为重链可变区，下划线部分依次为CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2、CDR-H3)
EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGKTFPLTFGQGTKLEIK(SEQ ID No.75)
QVQLQESGPGLVKPSETLSLTCTVSGFSLEDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(SEQ ID No.69)
实施例4
噬菌体结合ELISA筛选：
噬菌体的制备与纯化：挑取菌落克隆于4ml 2YT培养基中，37度200rpm过夜培养；以1：100转接至500ml 2xYT培养基中，37度200rpm培养至对数生长期；加入M13K07辅助噬菌体(MOI＝20)，37度静置孵育30分钟后，37度200rpm孵育30分钟；离心去掉上清，菌体重悬至500ml 2YT/Kan/Amp抗性培养基中培养过夜；将培养过夜的上清用1/5体积PEG/NaCl溶液沉淀静置1小时离心，将沉淀重悬于含30％丙三醇PBS溶液，4℃储存。将噬菌体溶液稀释10倍测定A268，均稀释至A268＝1，做2倍梯度稀释用于ELISA实验。
噬菌体结合ELISA实验：将1ug/ml Avidin包被96孔板过夜，用PBST洗2次，将100ul1ug/ml CD19-biotin加入Avidin包被的96孔板，孵育1小时后，用PBST洗2次后，加入梯度稀释的噬菌体孵育1小时，PBST洗4次，加入1:5000稀释抗M13噬菌体-HRP抗体100ul孵育30分钟，PBST洗4次后，加入TMB底物反应10分钟，加入1M硫酸溶液终止反应，读取OD450nm光吸收。具体结果如表4、表5、图4、图5所示，其中，EC50越小代表，达到同样的饱和信号所需相应克隆稀释倍数越大，即相应克隆结合能力越强。
表4
| 克隆-FMC63突变库 | EC50(10-2-A268) |
| 4B10 | 3.92 |
| 8A10 | 0.14 |
| FMC63 | 9.62 |
| NC | 25.00 |
表5
| 克隆-C2146突变库 | EC50(10-2-A268) |
| 7F12 | 1.91 |
| 7C3 | 1.32 |
| 7E11 | 0.49 |
| C2146 | 2.83 |
| NC | ND |
ND:Not Determined
实施例5
噬菌体竞争ELISA筛选：
将CDR突变重复较多的TG1克隆涂板(4B10及8A10)，挑取单克隆，IPTG诱导后纯化phage，纯化的phage经浓度测定后，稀释梯度(3倍稀释)与3ug/ml FMC63mIgG2a竞争结合CD19，检测使用抗鼠Fc二抗，以此比较不同克隆的IC50及Emax，具体检测结果如表6和图6所示。
表6
| 克隆号 | IC50(A268) | Emax(最大抑制率) |
| 4B11 | 1.64 | 56％ |
| 8A10 | 1.03 | 75％ |
| FMC63 | >5.4 | 20％ |
| NC | >5.4 | 19％ |
IC50越小代表相应克隆的单链抗体竞争FMC63mIgG2a至50％结合能力越强；Emax(最大抑制率)越大代表相应克隆的单链抗体相同A268浓度下竞争能力越强。
以上结果表明，4B10和8A10克隆能够竞争FMC63mIgG2a结合CD19的位点，提示4B10和8A10克隆结合CD19epitope与FMC63相同。
实施例6
FMC63 scFv突变体Koff测定：
将FMC63 scFv突变体(4B10，8A10)转接到pGCIgGH1(吉凯基因)(载体序列如SEQID No.76所示)，构建可以表达分泌型FMC63 scFv突变体hIgG1Fc融合蛋白，利用293真核表达系统瞬时表达96小时后，培养基上清中的scFv-hIgG1利用proteinA column进行纯化。
利用BLI技术测定Koff：将CD19-His抗原通过氨基偶联至AR2G芯片(Fortie Bio)，scFv-hIgG1突变体作为流动相，结合实践为120秒，解离时间为180秒，利用Fortie Bio系统测定，数据用Octet分析软件分析，Local法拟合动力学曲线，计算scFv-hIgG1突变体与CD19-His之间的解离常数。
表7
| 克隆(scFv-hIgG1) | Koff(1/s) | Response | Full R2 |
| 4B10 | 8.95x10-4 | 0.3498 | 0.974 |
| 8A10 | 9.13x10-4 | 0.4178 | 0.990 |
| FMC63 | 1.06x10-3 | 0.4658 | 0.969 |
| NC | ND | 0.09 | 0.967 |
以上结果显示，4B10及8A10克隆显示出较小的Koff值。
载体pGCIgGH1全序列：
AGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAAGATCTCTAGAAGCTGGGTACCTTGTGCCCGGGCGCCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTCTTAAGGGTGTCCAGTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCGGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGATACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGAGGTCGACTCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGCATATACAGTCAGCATATGATACCCAGTAGTAGAGTGGGAGTGCTATCCTTTGCATATGCCGCCACCTCCCAAGGGGGCGTGAATTTTCGCTGCTTGTCCTTTTCCTGCTGCTTATCGATGATAAGCTGTCAAACATGAGAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCCTGCTTCATCCCCGTGGCCCGTTGCTCGCGTTTGCTGGCGGTGTCACTGGCCCCGTGGGTTAGGGACGGGGTCCCCCATGGGGAATGGTTTATGGTTCGTGGGGGTTATTATTTTGGGCGTTGCGTGGGGTCAGGTCCACGACTGGACTGAGCAGACAGACCCATGGTTTTTGGATGGCCTGGGCATGGACCGCATGTACTGGCGCGACACGAACACCGGGCGTCTGTGGCTGCCAAACACCCCCGACCCCCAAAAACCACCGCGCGGATTTCTGGCGTGCCAAGCTAGTCGACCAATTCTCATGTTTGACAGCTTATCATCGCAGATCCGGGCAACGTTGTTGCCATTGCTGCAGGCGCAGAACTGGTAGGTATGGAAGATCT(SEQ ID No.76)
实施例7
FMC63 scFv突变体及C2146突变体细胞结合实验：
将FMC63突变体和C2146突变体scFv，分别各自转接到pGCIgGH1质粒(吉凯基因)，构建可以表达分泌型scFv-hIgG1Fc融合蛋白，利用293真核表达系统瞬时表达96小时后，培养基上清中的scFv-hIgG1利用proteinA column进行纯化。
表8
将Raji细胞或K562-CD19细胞(CD19+)培养于含10％FBS 1640培养基中，离心后用含1％BSA的PBS溶液重悬至5x106/ml，分至1.5ml EP管中，每管100ul，加入以上scFv-hIgG1做梯度稀释终浓度为50ug/ml，5ug/ml，0.5ug/ml，0.05ug/ml，0.005ug/ml。反应体系于4度孵育1小时后，用冰冷的PBS洗2次后加入抗人IgG Fc-PE标记二抗于4度孵育30min，用冰冷的PBS洗2次后用PBS溶液重悬，读取FACS。以荧光中位值(MFI)为纵坐标，scFv-hIgG1浓度为横坐标计算结合EC50。FMC63，4B10，8A10及C21hIgG1与Raji细胞结合结果如图7所示，C2146，7F12，7C3及7E11 hIgG1与K562-CD19细胞结合结果如图8所示，对应计算获得的各克隆与Raji或K562-CD19细胞结合的EC50结果如表9和表10所示。
表9
| FMC63突变库克隆-hIgG1 | Raji细胞结合EC50ug/ml |
| 4B10-hIgG1 | 0.16 |
| 8A10-hIgG1 | 0.13 |
| C21-hIgG1 | 0.09 |
| FMC63-hIgG1 | 0.20 |
表10
在同一次实验中，用组合克隆C21和7E11hIgG1与Raji细胞结合，并计算EC50，FMC63及C21hIgG1与Raji细胞结合结果如图9所示，C2146及7E11hIgG1与Raji细胞结合结果如图10所示，对应的EC50计算结果如表11所示。
表11
| 组合克隆 | Raji细胞结合EC50ug/ml |
| C21 | 0.23 |
| FMC63 | 1.08 |
| 7E11 | 0.36 |
| C2146 | 2.63 |
以上结果总结，FMC63突变库中C21克隆及C2146突变库中7E11克隆对CD19的亲和力显著提高(3-6倍)。
实施例8
FMC63突变体及C2146突变改造为嵌合抗原受体：
将FMC63突变体4B10，8A10，C21及C2146突变体7F12，7C3，7E11单链抗体，通过标准分子生物学方法链接BBz，序列如下，其中Hinge和CD137共刺激结构域用下划线标出，将构建完成的scFv-BBz通过BamHI和EcoRI酶切位点插入GV401载体(如图11所示)。BBz：CD8Hinge-CD8 TM-CD137-CD3ζ
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGC GTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTG TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA AGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID No.77)
实施例9
慢病毒包装：将包含scFv突变体BBz的GV401载体，与pCMV-VSVGenv、pCMV-Gag/pol两种质粒按照质量比3:2:2，利用磷酸钙沉淀法瞬时转染293T细胞，48-72小时后收集上清，此上清中含有VSVG包装的慢病毒颗粒。
T细胞的激活与感染：将分离自健康人外周血单核细胞(PBMC)，培养于含200IU/mlhIL2，10％FBS X-vivo15培养基中；利用抗CD3(OKT3克隆)及CD28抗体(15E8克隆)激活24小时后，加入含有FMC63突变体BBz及C2146突变体BBz的慢病毒，感染后72小时通过T细胞表达EGFP检测感染效率，具体结果如表12和表13所示。
表12
| FMC63突变库克隆 | 感染效率EGFP％ |
| 4B10-BBz | 35.5％ |
| 8A10-BBz | 50.9％ |
| C21-BBz | 61.4％ |
| FMC63-BBz | 37.5％ |
表13
| C2146突变库克隆 | 感染效率EGFP％ |
| 7F12-BBz | 45.2％ |
| 7C3-BBz | 52.6％ |
| 7E11-BBz | 48.2％ |
| C2146-BBz | 40.1％ |
细胞因子释放实验：将Raji细胞(CD19+)用含2％FBS的1640培养基重悬至105/ml，取U型底96孔板，加入100ul；将T细胞克隆稀释至105/ml后，每孔加入100ul，孵育过夜，去上清50ul，用BD FACS array测定如下细胞因子：IL2，IFNγ，TNFα，IL6等，通过与背景释放比值，计算细胞因子释放倍数变化，具体结果如表14和表15所示。
表14
表15
| C2146突变库克隆 | IL2倍数 | IFNγ倍数 | TNFα倍数 | IL6倍数 |
| 7F12-BBz | 163 | 105 | 51 | 1.6 |
| 7C3-BBz | 122 | 71 | 89 | 1.5 |
| 7E11-BBz | 207 | 115 | 120 | 1.7 |
| C2146-BBz | 157 | 87 | 96 | 1.4 |
细胞杀伤实验：将Raji细胞，用含2％FBS的1640培养基重悬至105/ml，取U型底96孔板，加入100ul；将T细胞克隆稀释至3x106/ml，按照E:T为30，10，3，1，0.3加入100ul，以只含有相应数量T细胞的孔作为对照，37度培养4小时，去50ul上清，用Promega LDH ReleaseKit测定肿瘤细胞裂解效率，具体结果如图12、图13、表16、表17所示。
表16
| FMC63突变库克隆 | EC50-杀伤 |
| 4B10-BBz | 6.7 |
| 8A10-BBz | 9.7 |
| C21-BBz | 4.4 |
| FMC63-BBz | 13.9 |
表17
| C2146突变库克隆 | EC50-杀伤 |
| 7F12-BBz | 8.9 |
| 7C3-BBz | 22.1 |
| 7E11-BBz | 6.7 |
| C2146-BBz | 12.2 |
EC50越小代表杀伤活性越高，以上结果显示组合突变C21和7E11克隆与单突变相比，细胞因子释放及杀伤效率更高。
实施例10
CART-7E11体内药效验证：
验证CART体内药效实验，文献与专利中所用荷瘤细胞为Raji，每只小鼠给予2x105细胞，在第4天给予3-5x106CART细胞。
为比较7E11克隆与C2146药效，本实验制备了NSG小鼠荷瘤晚期模型，细节如下：起始给予小鼠Raji细胞量为1x106，为文献报导的5倍；给予CART细胞的时间为第7天，相比文献报导晚了将近1倍时间；给予CART数量为1x106，为文献中报导的1/5。
制备Raji-ffluc细胞：ffluc(firefly Luciferase)基因，经标准分子生物学方法插入Ubc promoter-ffluc-IRES-PuroR读码框中，将读码框插入GV260载体(吉凯基因)并包装成慢病毒，通过感染Raji细胞，利用1ug/ml puromycin筛选后测试Luciferase表达。
NSG小鼠晚期荷瘤模型：第一天，将1x106Raji-ffluc细胞重悬于100ul PBS溶液；经尾静脉给予4-6周大NSG小鼠(NOD scid IL2Rγnull)，100ul/只。第七天，150mg/ml荧光素经腹腔给予小鼠，10分钟后检测荧光强度(PerkinElmer IVIS Spectrum)将100ul PBS、1x106CART-C2146和1x106CART-7E11经尾静脉分别给予小鼠(每组4只)。
第14天，小鼠活体成像定量结果表明在Raji荷瘤晚期模型中CART-7E11比CART-C2146药效更好，具体结果如图14所示。
NSG小鼠外周血细胞因子释放检测：
每只小鼠经尾静脉取血40ul(肝素抗凝)，用40ul PBS溶液重悬，1500rpm离心5分钟后，取上清，用BD FACS array方法，检测外周血中IFNγ释放。
第14天，小鼠外周血IFNγ释放检测结果表明，CART-7E11在小鼠体内释放了更强的细胞因子，提示更强的杀伤和扩增活性，具体结果如图15所示。
综上所述，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
序列表
<110> 上海吉凯基因化学技术有限公司
<120> 一种抗CD19抗体及其制备方法和用途
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acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 120
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agtcaggaca ttagtaaata tttaaattgg tatcagcaga aaccagatgg aactgttaaa 540
ctcctgatct accatacatc aagattacac tcaggagtcc catcaaggtt cagtggcagt 600
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<211> 242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile 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 Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser
130 135 140
Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
145 150 155 160
Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
165 170 175
Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu
195 200 205
Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln
210 215 220
Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
225 230 235 240
Ile Thr
<210> 11
<211> 726
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gaaattgtga tgacccagtc acccgccact cttagccttt cacccggtga gcgcgcaacc 60
ctgtcttgca gagcctccca agacatctca aaatacctta attggtatca acagaagccc 120
ggacaggctc ctcgccttct gatctaccac accagccggc tccattctgg aatccctgcc 180
aggttcagcg gtagcggatc tgggaccgac tacaccctca ctatcagctc actgcagcca 240
gaggacttcg ctgtctattt ctgtcagcaa gggaacaccc tgccctacac ctttggacag 300
ggcaccaagc tcgagattaa aggtggaggt ggcagcggag gaggtgggtc cggcggtgga 360
ggaagccagg tccaactcca agaaagcgga ccgggtcttg tgaagccatc agaaactctt 420
tcactgactt gtactgtgag cggagtgtct ctccccgatt acggggtgtc ttggatcaga 480
cagccaccgg ggaagggtct ggaatggatt ggagtgattt ggggctctga gactacttac 540
tacaattcat ccctcaagtc acgcgtcacc atctcaaagg acaactctaa gaatcaggtg 600
tcactgaaac tgtcatctgt gaccgcagcc gacaccgccg tgtactattg cgctaagcat 660
tactattatg gcgggagcta cgcaatggat tactggggac agggtactct ggtcaccgtg 720
tccagc 726
<210> 11
<211> 242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala 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 Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
130 135 140
Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser
180 185 190
Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr
195 200 205
Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 12
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
1 5 10
<210> 13
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser
1 5 10 15
<210> 14
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
1 5 10
<210> 15
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn
1 5 10
<210> 16
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
His Thr Ser Arg Leu His Ser
1 5
<210> 17
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Gln Gln Gly Asn Thr Leu Pro Tyr Thr
1 5
<210> 18
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
1 5 10
<210> 19
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser
1 5 10 15
<210> 20
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
1 5 10
<210> 21
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn
1 5 10
<210> 22
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
His Thr Ser Arg Leu His Ser
1 5
<210> 23
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Gln Gln Gly Asn Thr Leu Pro Tyr Thr
1 5
<210> 24
<211> 67
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tcacatgcac tgtctcaggg gtctcattac ccgactatgg tgtaagctgg attcgccagc 60
ctccacg 67
<210> 25
<211> 79
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
agggtctgga gtggctggga gtaatatggg gtagtgaaac cacatactat aattcagctc 60
tcaaatccag actgaccat 79
<210> 26
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg gactactggg 60
gccaaggaac ct 72
<210> 27
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
acagagtcac catcagttgc agggcaagtc aggacattag taaatattta aattggtatc 60
agcagaaacc ag 72
<210> 28
<211> 61
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca tcaaggttca 60
g 61
<210> 29
<211> 64
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga ggggggacca 60
agct 64
<210> 30
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ccctcatagt tagcgtaacg 20
<210> 31
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
agcggataac aatttcacac agga 24
<210> 32
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
ccctgagaca gtgcatgtga 20
<210> 33
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
tcccagccac tccagaccct 20
<210> 34
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tttggcacag tagtaaatgg 20
<210> 35
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gcaactgatg gtgactctgt 20
<210> 36
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gtagatcagg agtttaacag 20
<210> 37
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
ttggcaaaag taagtggcaa 20
<210> 38
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ctgacttgta ctgtgagcgg agtgtctctc cccgattacg gggtgtcttg gatcagacag 60
ccaccg 66
<210> 39
<211> 79
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
agggtctgga atggattgga gtgatttggg gctctgagac tacttactac aattcatccc 60
tcaagtcacg cgtcaccat 79
<210> 40
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
cgtgtactat tgcgctaagc attactatta tggcgggagc tacgcaatgg attactgggg 60
acagggtact 70
<210> 41
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
ccctgtcttg cagagcctcc caagacatct caaaatacct taattggtat caacag 56
<210> 42
<211> 61
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct gccaggttca 60
g 61
<210> 43
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
ctgtctattt ctgtcagcaa gggaacaccc tgccctacac ctttggacag ggcaccaag 59
<210> 44
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
ccctcatagt tagcgtaacg 20
<210> 45
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
agcggataac aatttcacac agga 24
<210> 46
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
tccgctcaca gtacaagtca g 21
<210> 47
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
tccaatccat tccagaccct 20
<210> 48
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
cttagcgcaa tagtacacgg 20
<210> 49
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
ggaggctctg caagacaggg 20
<210> 50
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
gtagatcaga aggcgaggag 20
<210> 51
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
ttgctgacag aaatagacag 20
<210> 52
<211> 726
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60
acatgcactg tctcagggtt ttcattagag gactatggtg taagctggat tcgccagcct 120
ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180
tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240
aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300
tacggtggta gctatgctat ggactactgg ggccaaggaa cctcagtcac cgtctcctca 360
ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcggacat ccagatgaca 420
cagactacat cctccctgtc tgcctctctg ggagacagag tcaccatcag ttgcagggca 480
agtcaggaca ttagtaaata tttaaattgg tatcagcaga aaccagatgg aactgttaaa 540
ctcctgatct accatacatc aagattacac tcaggagtcc catcaaggtt cagtggcagt 600
gggtctggaa cagattattc tctcaccatt agcaacctgg agcaagaaga tattgccact 660
tacttttgcc aacagggtaa tacgcttccg tacacgttcg gaggggggac caagctggag 720
atcaca 726
<210> 53
<211> 726
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60
acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 120
ccacgaaagg gtccggagtg gctgggagta atatggggta gtgaaaccac atactataat 180
tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240
aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300
tacggtggta gctatgctat ggactactgg ggccaaggaa cctcagtcac cgtctcctca 360
ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcggacat ccagatgaca 420
cagactacat cctccctgtc tgcctctctg ggagacagag tcaccatcag ttgcagggca 480
agtcaggaca ttagtaaata tttaaattgg tatcagcaga aaccagatgg aactgttaaa 540
ctcctgatct accatacatc aagattacac tcaggagtcc catcaaggtt cagtggcagt 600
gggtctggaa cagattattc tctcaccatt agcaacctgg agcaagaaga tattgccact 660
tacttttgcc aacagggtaa gacgtttccg cttacgttcg gaggggggac caagctggag 720
atcaca 726
<210> 54
<211> 726
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60
acatgcactg tctcagggtt ttcattagag gactatggtg taagctggat tcgccagcct 120
ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180
tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240
aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300
tacggtggta gctatgctat ggactactgg ggccaaggaa cctcagtcac cgtctcctca 360
ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcggacat ccagatgaca 420
cagactacat cctccctgtc tgcctctctg ggagacagag tcaccatcag ttgcagggca 480
agtcaggaca ttagtaaata tttaaattgg tatcagcaga aaccagatgg aactgttaaa 540
ctcctgatct accatacatc aagattacac tcaggagtcc catcaaggtt cagtggcagt 600
gggtctggaa cagattattc tctcaccatt agcaacctgg agcaagaaga tattgccact 660
tacttttgcc aacagggtaa gacgtttccg cttacgttcg gaggggggac caagctggag 720
atcaca 726
<210> 55
<211> 726
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
gaaattgtga tgacccagtc acccgccact cttagccttt cacccggtga gcgcgcaacc 60
ctgtcttgca gagcctccca agacatctca aaatacctta attggtatca acagaagccc 120
ggacaggctc ctcgccttct gatctaccac accagccggc tccattctgg aatccctgcc 180
aggttcagcg gtagcggatc tgggaccgac tacaccctca ctatcagctc actgcagcca 240
gaggacttcg ctgtctattt ctgtcagcaa gggaacaccc tgccctacac ctttggacag 300
ggcaccaagc tcgagattaa aggtggaggt ggcagcggag gaggtgggtc cggcggtgga 360
ggaagccagg tccaactcca agaaagcgga ccgggtcttg tgaagccatc agaaactctt 420
tcactgactt gtactgtgag cggattttct ctcgaggatt acggggtgtc ttggatcaga 480
cagccaccgg ggaagggtct ggaatggatt ggagtgattt ggggctctga gactacttac 540
tacaattcat ccctcaagtc acgcgtcacc atctcaaagg acaactctaa gaatcaggtg 600
tcactgaaac tgtcatctgt gaccgcagcc gacaccgccg tgtactattg cgctaagcat 660
tactattatg gcgggagcta cgcaatggat tactggggac agggtactct ggtcaccgtg 720
tccagc 726
<210> 56
<211> 726
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
gaaattgtga tgacccagtc acccgccact cttagccttt cacccggtga gcgcgcaacc 60
ctgtcttgca gagcctccca agacatctca aaatacctta attggtatca acagaagccc 120
ggacaggctc ctcgccttct gatctaccac accagccggc tccattctgg aatccctgcc 180
aggttcagcg gtagcggatc tgggaccgac tacaccctca ctatcagctc actgcagcca 240
gaggacttcg ctgtctattt ctgtcagcaa gggaagacct ttccccttac ctttggacag 300
ggcaccaagc tcgagattaa aggtggaggt ggcagcggag gaggtgggtc cggcggtgga 360
ggaagccagg tccaactcca agaaagcgga ccgggtcttg tgaagccatc agaaactctt 420
tcactgactt gtactgtgag cggagtgtct ctccccgatt acggggtgtc ttggatcaga 480
cagccaccgg ggaagggtct ggaatggatt ggagtgattt ggggctctga gactacttac 540
tacaattcat ccctcaagtc acgcgtcacc atctcaaagg acaactctaa gaatcaggtg 600
tcactgaaac tgtcatctgt gaccgcagcc gacaccgccg tgtactattg cgctaagcat 660
tactattatg gcgggagcta cgcaatggat tactggggac agggtactct ggtcaccgtg 720
tccagc 726
<210> 57
<211> 726
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
gaaattgtga tgacccagtc acccgccact cttagccttt cacccggtga gcgcgcaacc 60
ctgtcttgca gagcctccca agacatctca aaatacctta attggtatca acagaagccc 120
ggacaggctc ctcgccttct gatctaccac accagccggc tccattctgg aatccctgcc 180
aggttcagcg gtagcggatc tgggaccgac tacaccctca ctatcagctc actgcagcca 240
gaggacttcg ctgtctattt ctgtcagcaa gggaagacct ttccccttac ctttggacag 300
ggcaccaagc tcgagattaa aggtggaggt ggcagcggag gaggtgggtc cggcggtgga 360
ggaagccagg tccaactcca agaaagcgga ccgggtcttg tgaagccatc agaaactctt 420
tcactgactt gtactgtgag cggattttct ctcgaggatt acggggtgtc ttggatcaga 480
cagccaccgg ggaagggtct ggaatggatt ggagtgattt ggggctctga gactacttac 540
tacaattcat ccctcaagtc acgcgtcacc atctcaaagg acaactctaa gaatcaggtg 600
tcactgaaac tgtcatctgt gaccgcagcc gacaccgccg tgtactattg cgctaagcat 660
tactattatg gcgggagcta cgcaatggat tactggggac agggtactct ggtcaccgtg 720
tccagc 726
<210> 58
<211> 242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 58
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Phe Ser Leu Glu Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile 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 Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser
130 135 140
Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
145 150 155 160
Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
165 170 175
Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu
195 200 205
Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln
210 215 220
Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
225 230 235 240
Ile Thr
<210> 59
<211> 242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 59
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Pro Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile 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 Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser
130 135 140
Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
145 150 155 160
Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
165 170 175
Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu
195 200 205
Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln
210 215 220
Gln Gly Lys Thr Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu
225 230 235 240
Ile Thr
<210> 60
<211> 242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 60
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Phe Ser Leu Glu Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile 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 Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser
130 135 140
Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
145 150 155 160
Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
165 170 175
Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu
195 200 205
Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln
210 215 220
Gln Gly Lys Thr Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu
225 230 235 240
Ile Thr
<210> 61
<211> 242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 61
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala 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 Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
130 135 140
Thr Val Ser Gly Phe Ser Leu Glu Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser
180 185 190
Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr
195 200 205
Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 62
<211> 242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 62
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala 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 Val Tyr Phe Cys Gln Gln Gly Lys Thr Phe Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
130 135 140
Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser
180 185 190
Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr
195 200 205
Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 63
<211> 242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 63
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala 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 Val Tyr Phe Cys Gln Gln Gly Lys Thr Phe Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
130 135 140
Thr Val Ser Gly Phe Ser Leu Glu Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser
180 185 190
Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr
195 200 205
Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 64
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 64
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Phe Ser Leu Glu Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile 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 Thr Ser Val Thr Val Ser Ser
115 120
<210> 65
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 65
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Pro Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile 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 Thr Ser Val Thr Val Ser Ser
115 120
<210> 66
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 66
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Phe Ser Leu Glu Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile 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 Thr Ser Val Thr Val Ser Ser
115 120
<210> 67
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 67
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Glu Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 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 Thr Leu Val Thr Val Ser Ser
115 120
<210> 68
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 68
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
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 Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 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 Thr Leu Val Thr Val Ser Ser
115 120
<210> 69
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 69
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Glu Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 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 Thr Leu Val Thr Val Ser Ser
115 120
<210> 70
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 70
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys 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 Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
100 105
<210> 71
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 71
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys 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 Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Phe Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
100 105
<210> 72
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 72
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys 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 Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Phe Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
100 105
<210> 73
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 73
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala 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 Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 74
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 74
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala 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 Val Tyr Phe Cys Gln Gln Gly Lys Thr Phe Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 75
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 75
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala 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 Val Tyr Phe Cys Gln Gln Gly Lys Thr Phe Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 76
<211> 7252
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 76
agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga 60
gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa 120
ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta 180
tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca 240
ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt 300
gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg 360
aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt 420
tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg 480
tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct 540
ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt 600
ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg 660
gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc 720
tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg 780
gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc 840
aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag 900
ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag 960
gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt 1020
ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca 1080
cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa 1140
gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaagatctct 1200
agaagctggg taccttgtgc ccgggcgcca ccatggagtt tgggctgagc tggctttttc 1260
ttgtcgcgat tcttaagggt gtccagtgcg acaaaactca cacatgccca ccgtgcccag 1320
cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc 1380
tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc 1440
ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc 1500
cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc 1560
aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc ctcccagccc 1620
ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag gtgtacaccc 1680
tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc ctggtcaaag 1740
gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact 1800
acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac agcaagctca 1860
ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg atgcatgagg 1920
ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa tgagcggccg 1980
ctcgaggccg gcaaggccgg atccagacat gataagatac attgatgagt ttggacaaac 2040
cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 2100
atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 2160
gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg 2220
tggtatggct gattatgatc cggctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc 2280
tgacacatgc agctcccgga tacggtcaca gcttgtctgt aagcggatgc cgggagcaga 2340
caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgaggtcg 2400
actctagagg atcgatcccc gccccggacg aactaaacct gactacgaca tctctgcccc 2460
ttcttcgcgg ggcagtgcat gtaatccctt cagttggttg gtacaacttg ccaactgggc 2520
cctgttccac atgtgacacg gggggggacc aaacacaaag gggttctctg actgtagttg 2580
acatccttat aaatggatgt gcacatttgc caacactgag tggctttcat cctggagcag 2640
actttgcagt ctgtggactg caacacaaca ttgcctttat gtgtaactct tggctgaagc 2700
tcttacacca atgctggggg acatgtacct cccaggggcc caggaagact acgggaggct 2760
acaccaacgt caatcagagg ggcctgtgta gctaccgata agcggaccct caagagggca 2820
ttagcaatag tgtttataag gcccccttgt taaccctaaa cgggtagcat atgcttcccg 2880
ggtagtagta tatactatcc agactaaccc taattcaata gcatatgtta cccaacggga 2940
agcatatgct atcgaattag ggttagtaaa agggtcctaa ggaacagcga tatctcccac 3000
cccatgagct gtcacggttt tatttacatg gggtcaggat tccacgaggg tagtgaacca 3060
ttttagtcac aagggcagtg gctgaagatc aaggagcggg cagtgaactc tcctgaatct 3120
tcgcctgctt cttcattctc cttcgtttag ctaatagaat aactgctgag ttgtgaacag 3180
taaggtgtat gtgaggtgct cgaaaacaag gtttcaggtg acgcccccag aataaaattt 3240
ggacgggggg ttcagtggtg gcattgtgct atgacaccaa tataaccctc acaaacccct 3300
tgggcaataa atactagtgt aggaatgaaa cattctgaat atctttaaca atagaaatcc 3360
atggggtggg gacaagccgt aaagactgga tgtccatctc acacgaattt atggctatgg 3420
gcaacacata atcctagtgc aatatgatac tggggttatt aagatgtgtc ccaggcaggg 3480
accaagacag gtgaaccatg ttgttacact ctatttgtaa caaggggaaa gagagtggac 3540
gccgacagca gcggactcca ctggttgtct ctaacacccc cgaaaattaa acggggctcc 3600
acgccaatgg ggcccataaa caaagacaag tggccactct tttttttgaa attgtggagt 3660
gggggcacgc gtcagccccc acacgccgcc ctgcggtttt ggactgtaaa ataagggtgt 3720
aataacttgg ctgattgtaa ccccgctaac cactgcggtc aaaccacttg cccacaaaac 3780
cactaatggc accccgggga atacctgcat aagtaggtgg gcgggccaag ataggggcgc 3840
gattgctgcg atctggagga caaattacac acacttgcgc ctgagcgcca agcacagggt 3900
tgttggtcct catattcacg aggtcgctga gagcacggtg ggctaatgtt gccatgggta 3960
gcatatacta cccaaatatc tggatagcat atgctatcct aatctatatc tgggtagcat 4020
aggctatcct aatctatatc tgggtagcat atgctatcct aatctatatc tgggtagtat 4080
atgctatcct aatttatatc tgggtagcat aggctatcct aatctatatc tgggtagcat 4140
atgctatcct aatctatatc tgggtagtat atgctatcct aatctgtatc cgggtagcat 4200
atgctatcct aatagagatt agggtagtat atgctatcct aatttatatc tgggtagcat 4260
atactaccca aatatctgga tagcatatgc tatcctaatc tatatctggg tagcatatgc 4320
tatcctaatc tatatctggg tagcataggc tatcctaatc tatatctggg tagcatatgc 4380
tatcctaatc tatatctggg tagtatatgc tatcctaatt tatatctggg tagcataggc 4440
tatcctaatc tatatctggg tagcatatgc tatcctaatc tatatctggg tagtatatgc 4500
tatcctaatc tgtatccggg tagcatatgc tatcctcatg catatacagt cagcatatga 4560
tacccagtag tagagtggga gtgctatcct ttgcatatgc cgccacctcc caagggggcg 4620
tgaattttcg ctgcttgtcc ttttcctgct gcttatcgat gataagctgt caaacatgag 4680
aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 4740
ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 4800
tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 4860
atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 4920
attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 4980
gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 5040
agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 5100
aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 5160
cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 5220
cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 5280
actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 5340
cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 5400
ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 5460
ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 5520
gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 5580
gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 5640
ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 5700
cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 5760
caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 5820
taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 5880
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 5940
cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 6000
gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 6060
aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 6120
cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 6180
tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 6240
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 6300
ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 6360
ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 6420
tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 6480
tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 6540
ctggcctttt gctggccttt tgctcacatg aagctgtccc tgatggtcgt catctacctg 6600
cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 6660
aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 6720
ggccccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc 6780
caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga 6840
gtggtgaatc cgttagcgag gtgccgccct gcttcatccc cgtggcccgt tgctcgcgtt 6900
tgctggcggt gtcactggcc ccgtgggtta gggacggggt cccccatggg gaatggttta 6960
tggttcgtgg gggttattat tttgggcgtt gcgtggggtc aggtccacga ctggactgag 7020
cagacagacc catggttttt ggatggcctg ggcatggacc gcatgtactg gcgcgacacg 7080
aacaccgggc gtctgtggct gccaaacacc cccgaccccc aaaaaccacc gcgcggattt 7140
ctggcgtgcc aagctagtcg accaattctc atgtttgaca gcttatcatc gcagatccgg 7200
gcaacgttgt tgccattgct gcaggcgcag aactggtagg tatggaagat ct 7252
<210> 77
<211> 672
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 77
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180
ctgtcactgg ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc 240
aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 300
tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 360
gcccccgcgt acaagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 420
gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 480
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 540
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 600
taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 660
ccccctcgct aa 672
Claims (11)
1.一种抗CD19抗体，所述抗CD19抗体包括重链可变区和轻链可变区，所述抗CD19抗体具有如下技术特征中的一个或多个：
<1>重链可变区的CDR包括氨基酸序列如SEQ ID No.1所示的CDR-H1；
<2>重链可变区的CDR包括氨基酸序列如SEQ ID No.2或SEQ ID No.3所示的CDR-H2；
<3>重链可变区的CDR包括氨基酸序列如SEQ ID No.4所示的CDR-H3；
<4>轻链可变区的CDR包括氨基酸序列如SEQ ID No.5所示的CDR-L1；
<5>轻链可变区的CDR包括氨基酸序列如SEQ ID No.6所示的CDR-L2；
<6>轻链可变区的CDR包括氨基酸序列如SEQ ID No.7所示的CDR-L3。
2.如权利要求1所述的一种抗CD19抗体，其特征在于，所述抗CD19抗体的重链可变区的CDR包括氨基酸序列如SEQ ID No.1所示的CDR-H1、氨基酸序列如SEQ ID No.2或SEQ IDNo.3所示的CDR-H2和氨基酸序列如SEQ ID No.4所示的CDR-H3；
和/或，所述抗CD19抗体的轻链可变区的CDR包括氨基酸序列如SEQ ID No.5所示的CDR-L1、氨基酸序列如SEQ ID No.6所示的CDR-L2和氨基酸序列如SEQ ID No.7所示的CDR-L3；
和/或，所述抗CD19抗体为单克隆抗体；
和/或，所述抗CD19抗体为单链抗体；
和/或，所述抗CD19抗体的重链可变区的氨基酸序列包括：
a)如SEQ ID No.64-69其中之一所示的氨基酸序列；或
b)与SEQ ID No.64-69其中之一所示的氨基酸序列具有80％以上同源性、且具有a)所限定的氨基酸序列功能的氨基酸序列；
和/或，所述抗CD19抗体的轻链可变区的氨基酸序列包括：
c)如SEQ ID No.70-75其中之一所示的氨基酸序列；或
d)与SEQ ID No.70-75其中之一所示的氨基酸序列具有80％以上同源性、且具有c)所限定的氨基酸序列功能的氨基酸序列。
3.一种分离的多核苷酸，编码如权利要求1-2任一权利要求所述的抗CD19抗体的重链可变区和/或轻链可变区或全长氨基酸。
4.一种构建体，含有如权利要求3所述的分离的多核苷酸。
5.一种抗体的表达系统，所述表达系统含有如权利要求4任一权利要求所述的构建体或基因组中整合有外源的如权利要求3所述的多核苷酸。
6.如权利要求1-2任一权利要求所述的抗CD19抗体的制备方法，包括如下步骤：在适合表达所述抗体的条件下，培养如权利要求5所述的抗体的表达系统，从而表达出所述的抗体，纯化分离出所述的抗体。
7.如权利要求1-2任一权利要求所述的抗CD19抗体在制备或筛选治疗药物中的用途、或制备诊断药物中的用途。
8.一种分离的多肽，所述多肽包括跨膜域、胞内域和胞外域，所述胞外域包括如权利要求1-2任一权利要求所述的抗CD19抗体。
9.如权利要求8所述的多肽，其特征在于，所述多肽为嵌合抗原受体；
和/或，所述跨膜域包括CD8α、CD28、DAP10；
和/或，所述胞内域包括4-1BB、CD28、OX40、ICOS、CD3zeta、DAP10；
和/或，所述多肽自N端至C端依次包括所述抗CD19抗体、跨膜域、胞内域。
10.一种细胞，所述细胞含膜结合的如权利要求8-9任一权利要求所述的多肽，所述细胞为T淋巴细胞和/或NK细胞；
和/或，所述多肽为嵌合抗原受体。
和/或，当所述多肽结合于CD19抗原时，所述T淋巴细胞和/或NK细胞可以活化和/或刺激从而得以增殖。
和/或，所述T淋巴细胞和/或NK细胞表面表达所述抗CD19抗体。
11.一种诊断试剂盒，包含诊断有效剂量的如权利要求1-2任一权利要求所述的抗CD19抗体或其免疫偶联物。
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