CN112480253A - 一种抗pd-l1纳米抗体及其衍生物和用途 - Google Patents
一种抗pd-l1纳米抗体及其衍生物和用途 Download PDFInfo
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Abstract
本发明涉及一种抗PD‑L1纳米抗体及其衍生物和用途。具体地,本发明提供了一种抗PD‑L1的纳米抗体,该抗体具有阻断PD‑L1与受体PD‑1结合的功能。并且,本发明提供了所述编码该纳米抗体的基因序列、相应的表达载体和能够表达该纳米抗体的宿主细胞,以及本发明纳米抗体的生产方法。同时还提供了同时具有本发明PD‑L1纳米抗体和免疫调节分子部分的融合蛋白,其可以在靶向中和肿瘤微环境的TGF‑β基础上抑制PD‑1/PD‑L1通路,可以使T细胞恢复活性,增强免疫应答,更有效地提高抑制肿瘤发生和发展的效果。
Description
技术领域
本发明涉及生物医学或生物制药技术领域,更具体地涉及一种抗PD-L1纳米抗体及其衍生物和用途。
背景技术
程序性死亡因子1配体1(programmed death 1 ligand 1,PD-L1)又称CD274,为B7家族成员,是PD-1的配体。PD-L1属于I型跨膜蛋白,共290个氨基酸,包含1个IgV样区、1个IgC样区、1个跨膜疏水区和1个由30个氨基酸组成的胞内区。
与其他B7家族分子不同的是,PD-L1具有负向调节免疫应答的作用。研究发现,PD-L1主要表达于活化的T细胞、B细胞、巨噬细胞和树突状细胞等,除淋巴细胞外,PD-L1也表达于其他多种组织如胸腺、心脏、胎盘等的内皮细胞,以及各类非淋巴系如黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌、头颈癌等。PD-L1在调节自身反应性T、B细胞和免疫耐受方面具有一定广泛性,并且在外周组织T和B细胞应答起作用。PD-L1在肿瘤细胞上的高表达与癌症患者的不良预后相关。
与PD-L1相结合的程序性死亡因子1(programmed death-1,PD-1)又称CD279,是CD28家族成员,其胞质区含有2个酪氨酸残基,靠近N端的1个位于免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitory motif,ITIM)中,靠近C端的1个位于免疫受体酪氨酸转化基序(immunoreceptor tyrosine-based switch motif,ITSM)中。PD-1主要表达在活化的T淋巴细胞、B淋巴细胞和巨噬细胞表面。在正常情况下,PD-1能够抑制T淋巴细胞的功能,促进Treg的功能,从而抑制自身免疫应答,防止自身免疫性疾病的发生。但在肿瘤的发生中,肿瘤细胞表达的PD-L1与PD-1结合后却能通过对淋巴细胞的抑制性作用促进肿瘤的免疫逃逸。PD-L1与PD-1的结合可导致多种生物学变化,引起免疫调控,如能够抑制淋巴细胞的增殖和活化、抑制CD4+T细胞向Th1和Th17细胞分化、抑制炎性细胞因子的释放等。
单克隆抗体在癌症的检测及生物靶向治疗方面成功的应用,引起了肿瘤治疗的变革。然而,传统的单抗(150kD)分子质量过大,难穿透组织,造成肿瘤区域的有效浓度较低,治疗效果不充分;传统的抗体具有很高的免疫原性,而改造的抗体很难达到原来的亲和力。此外,完全人源化的传统抗体开发周期长,生产成本高,稳定性不够等诸多因素限制其在临床中的应用及普及。
纳米抗体是目前最小的抗体分子,其分子量是普通抗体的1/10。纳米抗体除具备单克隆抗体的抗原反应性外,还拥有一些独特的功能特性,如分子质量小,稳定性强、可溶性好、易表达、免疫原性弱、穿透性强、靶向性强、人源化简单,制备成本低廉等,几乎完美克服了传统抗体开发周期长,稳定性较低,保存条件苛刻等缺陷。
然而,目前本领域尚缺乏令人满意的针对PD-L1的纳米抗体。因此,本领域迫切需要开发新的有效针对PD-L1的特异性纳米抗体。
发明内容
本发明的目的就是提供一类新的有效针对PD-L1的特异性纳米抗体。
在本发明的第一方面,提供了一种抗PD-L1纳米抗体VHH链的互补决定区CDR区,所述VHH链的互补决定区CDR由以下组成:
氨基酸序列如SEQ ID NO:5n+1所示的CDR1;
氨基酸序列如SEQ ID NO:5n+2所示的CDR2,或氨基酸序列与SEQ ID NO:2所示的序列具有大于85%的序列同一性的CDR2;和
氨基酸序列如SEQ ID NO:5n+3所示的CDR3;
其中,各n独立地为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14或15。
在另一优选例中,所述的n为0或1。
在另一优选例中,所述CDR2的氨基酸序列如SEQ ID NO:2、7、81、84、87、90、93或96所示。
在另一优选例中,所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
在本发明的第二方面,提供了一种抗PD-L1纳米抗体的VHH链,所述抗PD-L1纳米抗体的VHH链中包括如本发明第一方面所述的CDR1、CDR2和CDR3。
在另一优选例中,所述抗PD-L1纳米抗体的VHH链的氨基酸序列如SEQ ID NO:5n+4、82、85、88、91、94或97所示;
其中,所述n为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14或15;
其中,上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代1-8个(较佳地1-5个,更佳地1-3个)氨基酸残基,并能够保留PD-L1纳米抗体的PD-L1结合亲和力的衍生序列。
在另一优选例中,所述n为0或1。
在另一优选例中,所述抗PD-L1纳米抗体的VHH链的氨基酸序列如SEQ ID NO:4、9、82、85、88、91、94或97所示。
在本发明的第三方面,提供了一种抗PD-L1纳米抗体,所述抗PD-L1纳米抗体是针对PD-L1表位的纳米抗体,并且具有如本发明第二方面所述的抗PD-L1纳米抗体的VHH链。
在本发明的第四方面,提供了一种多核苷酸,所述多核苷酸编码选自下组的蛋白质:如本发明第一方面所述的抗PD-L1纳米抗体VHH链的CDR区、如本发明第二方面所述的抗PD-L1纳米抗体的VHH链,或如本发明第三方面所述的抗PD-L1纳米抗体。
在另一优选例中,所述多核苷酸具有如SEQ ID NO:5n、83、86、89、92、95或98所示的核苷酸序列;
其中,所述n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15或16。
在另一优选例中,所述的多核苷酸包括DNA或RNA。
在本发明的第五方面,提供了一种表达载体,所述表达载体含有如本发明第四方面所述的多核苷酸。
在另一优选例中,所述表达载体中还包含有编码免疫球蛋白的Fc段的核苷酸序列。
在另一优选例中,所述免疫球蛋白是IgG1、IgG2、IgG3、IgG4。
在本发明的第六方面,提供了一种宿主细胞,所述宿主细胞含有如本发明第五方面所述的表达载体,或其基因组中整合有如本发明第四方面所述的多核苷酸。
在另一优选例中,所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中,所述的宿主细胞选自下组:大肠杆菌、酵母细胞、哺乳动物细胞。
在本发明的第七方面,提供了一种产生抗PD-L1纳米抗体的方法,包括步骤:
(a)在适合产生纳米抗体的条件下,培养如本发明第六方面所述的宿主细胞,从而获得含所述抗PD-L1纳米抗体的培养物;以及
(b)从所述培养物中分离或回收所述的抗PD-L1纳米抗体。
在另一优选例中,所述的抗PD-L1纳米抗体具有如SEQ ID NO:5n+4、82、85、88、91、94或97所示的氨基酸序列;
其中,所述n为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14或15。
在本发明的第八方面,提供了一种纳米抗体融合蛋白,所述纳米抗体融合蛋白从N端到C端具有如式I所示的结构:
Z1-Z2-L-Z3 (式I)
式中,
Z1为如本发明第二方面所述的抗PD-L1纳米抗体的VHH链;
Z2为免疫球蛋白的Fc段;
L为接头序列;
Z3为免疫调节分子部分。
在另一优选例中,所述免疫球蛋白是IgG1、IgG2、IgG3、IgG4。
在另一优选例中,所述Z2的氨基酸序列如SEQ ID NO:99所示。
在另一优选例中,所述Z2的氨基酸序列与如SEQ ID NO:99所示的氨基酸序列相同或基本相同。
在另一优选例中,所述L具有选自下组的氨基酸序列:GGGGS、(GGGGS)2、(GGGGS)3、(GGGGS)4、(GGGGS)5,或其组合。
在另一优选例中,所述L的氨基酸序列如SEQ ID NO:100所示。
在另一优选例中,所述L的氨基酸序列与如SEQ ID NO:100所示的氨基酸序列相同或基本相同。
在另一优选例中,所述免疫调节分子为TGFβRII胞外结构域。
在另一优选例中,所述Z3的氨基酸序列如SEQ ID NO:101所示。
在另一优选例中,所述Z3的氨基酸序列与如SEQ ID NO:101所示的氨基酸序列相同或基本相同。
在另一优选例中,所述的基本相同是至多有50个(较佳地为1-20个,更佳地为1-10个、更佳地1-5个,最佳地为1-3个)氨基酸不相同,其中,所述的不相同包括氨基酸的取代、缺失或添加。
在另一优选例中,所述的基本相同是氨基酸序列与相应氨基酸序列的序列同一性至少为70%、至少为75%、至少为80%、至少为85%、至少为86%、至少为87%、至少为88%、至少为89%、至少为90%、至少为91%、至少为92%、至少为93%、至少为94%、至少为95%、至少为96%、至少为97%、至少为98%,或者至少为99%。
在另一优选例中,所述纳米抗体融合蛋白的氨基酸序列如SEQ ID NO:102所示。
在本发明的第九方面,提供了一种免疫偶联物,所述免疫偶联物含有:
(a)如本发明第二方面所述的抗PD-L1纳米抗体的VHH链、如本发明第三方面所述的抗PD-L1纳米抗体,或如本发明第八方面所述的纳米抗体融合蛋白;和
(b)选自下组的偶联部分:可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中,所述偶联部分为药物或毒素。
在另一优选例中,所述偶联部分为可检测标记物。
在另一优选例中,所述偶联物选自:荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如,DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如,顺铂)或任何形式的纳米颗粒等。
在另一优选例中,所述免疫偶联物含有:多价(如二价)的如本发明第二方面所述的抗PD-L1纳米抗体的VHH链、如本发明第三方面所述的抗PD-L1纳米抗体,或如本发明第八方面所述的纳米抗体融合蛋白。
在另一优选例中,所述多价是指,在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明第二方面所述的抗PD-L1纳米抗体的VHH链、如本发明第三方面所述的抗PD-L1纳米抗体,或如本发明第八方面所述的纳米抗体融合蛋白。
在本发明的第十方面,提供了如本发明第三方面所述的抗PD-L1纳米抗体或如本发明第八方面所述的纳米抗体融合蛋白的用途,用于制备(a)用于检测PD-L1分子的试剂;(b)用于治疗肿瘤的药物。
在另一优选例中,所述的检测包括流式检测、细胞免疫荧光检测。
在本发明的第十一方面,提供了一种药物组合物,包括:
(i)如本发明第一方面所述的抗PD-L1纳米抗体VHH链的互补决定区CDR、如本发明第二方面所述的抗PD-L1纳米抗体的VHH链、或如本发明第三方面所述的抗PD-L1纳米抗体、如本发明第八方面所述的纳米抗体融合蛋白,或如本发明第九方面所述的免疫偶联物;以及
(ii)药学上可接受的载体。
在另一优选例中,所述的药物组合物为注射剂型。
在另一优选例中,所述的药物组合物用于制备治疗肿瘤的药物,所述的肿瘤选自下组:胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、膀胱肿瘤,或其组合。
在本发明的第十二方面,提供了如本发明第三方面所述的抗PD-L1纳米抗体或如本发明第八方面所述的纳米抗体融合蛋白的一种或多种的用途:
(i)用于检测人PD-L1分子;
(ii)用于流式检测;
(iii)用于细胞免疫荧光检测;
(iv)用于治疗肿瘤;和
(v)用于肿瘤诊断。
在另一优选例中,所述用途为非诊断的和非治疗的。
在本发明的第十三方面,提供了一种重组蛋白,所述的重组蛋白具有:
(i)如本发明第二方面所述的重链可变区VHH的序列或如本发明第三方面所述的纳米抗体的序列,或如本发明第八方面所述的纳米抗体融合蛋白;以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中,所述的标签序列包括:6His标签、HA标签、Flag标签、Fc标签,或其组合。
在另一优选例中,所述的重组蛋白特异性结合于PD-L1蛋白。
在本发明的第十四方面,提供了如本发明第二方面所述的VHH链、如本发明第三方面所述的纳米抗体、如本发明第八方面所述的纳米抗体融合蛋白,或如本发明第九方面所述的免疫偶联物的用途,被用于制备药剂、试剂、检测板或试剂盒;
其中,所述试剂、检测板或试剂盒用于:检测样品中PD-L1蛋白;
其中,所述药剂用于治疗或预防表达PD-L1蛋白(即PD-L1阳性)的肿瘤。
在另一优选例中,所述肿瘤包括:胃癌、淋巴瘤、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、肾上腺肿瘤,或其组合。
在本发明的第十五方面,提供了一种检测样品中PD-L1蛋白的方法,包括步骤:
(1)将样品与如本发明第三方面所述的纳米抗体或如本发明第八方面所述的纳米抗体融合蛋白接触;
(2)检测是否形成抗原-抗体复合物,其中形成复合物就表示样品中存在PD-L1蛋白。
在另一优选例中,所述检测包括定性检测和定量检测。
在本发明的第十六方面,提供了一种治疗疾病的方法,所述方法包括,给需要的对象施用如本发明第三方面所述的纳米抗体、如本发明第八方面所述的纳米抗体融合蛋白或如本发明第九方面所述的免疫偶联物。
在另一优选例中,所述的对象包括哺乳动物。
在另一优选例中,所述哺乳动物为人。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
图1显示了本发明的纳米抗体可和细胞表面的人PD-L1蛋白结合,且部分抗体的结合效果和阳性对照相似。
图2显示了改造后的纳米抗体仍可和细胞表面的人PD-L1蛋白结合,且抗体的结合效果和阳性对照相似。
图3显示了改造后的纳米抗体仍可阻断PD-L1蛋白和细胞表面的人PD-1蛋白的结合,且抗体的阻断效果和阳性对照相似。
图4显示了本发明的纳米抗体可有效激活T细胞,且激活效果和阳性对照组抗体相似或更优。
图5显示了融合蛋白结构示意图。
图6显示了本发明的融合蛋白可和细胞表面的人PD-L1蛋白结合。
图7显示了本发明的融合蛋白可阻断PD-L1蛋白和细胞表面的人PD-1蛋白的结合,且抗体的阻断效果和阳性对照相似。
图8显示了本发明的融合蛋白可和TGFβ1、TGFβ2、TGFβ3结合。
图9显示了本发明的融合蛋白可有效阻断TGFβ/SMAD信号通路。
图10显示了本发明的融合蛋白可有效激活T细胞,且激活效果和阳性对照组抗体相似或更优。
图11显示了本发明的融合蛋白可以有效抑制小鼠体内的肿瘤生长。
具体实施方式
本发明人经过广泛而深入的研究,经过大量的筛选,首次开发了一类抗PD-L1纳米抗体。实验结果表明,本发明获得的PD-L1纳米抗体及其突变体衍生物能够有效阻断PD-L1与PD-1之间的相互作用,且具有较好的热稳定性。
具体地,本发明利用人源的PD-L1抗原蛋白免疫羊驼(Llama),获得高质量的免疫纳米抗体基因文库。其中,本发明人在所述免疫纳米抗体基因文库中意外地筛选人源化水平较高(序列同一性>85%)的纳米抗体基因序列。将PD-L1蛋白分子进行生物素化标记,并用酵母展示技术筛选免疫纳米抗体基因库,从而获得了PD-L1特异性的纳米抗体候选分子抗体基因。再将所获得的基因及其工程化突变体转至Expi-CHO细胞中,从抗体亲和力、阻断PD-L1与PD-1结合的能力、热稳定性、激活T细胞的活性等方面进行进一步的筛选,从而获得了一类能在体外高效表达的、且特异性高的纳米抗体株。
此外,实验结果表明,将本发明的纳米抗体序列(作为靶向部分)与IgG1 Fc片段(作为连接部分)以及TGFβRII胞外结构域(作为免疫调节分子部分)进行融合后所产生的融合蛋白,与PD-L1结果活性高,能够有效地阻断PD-L1与PD-1之间的相互作用,并且可有效阻断TGF-β/SMAD信号通路,能够有效地激活人T淋巴细胞,并且能够在小鼠体内有效地抑制肿瘤的生长。
在此基础上,完成了本发明。
本发明纳米抗体
如本文所用,术语“本发明纳米抗体”、“本发明的抗PD-L1纳米抗体”、“本发明PD-L1纳米抗体”可互换使用,均指特异性识别和结合于PD-L1(包括人PD-L1)的纳米抗体。特别优选的是VHH链的氨基酸序列如SEQ ID NO:4、9、82、85、88、91、94或97所示的纳米抗体。
如本文所用,术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白,其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连,而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH),其后是多个恒定区。每条轻链的一端有可变区(VL),另一端有恒定区;轻链的恒定区与重链的第一个恒定区相对,轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用,术语“单域抗体(VHH)”、“纳米抗体”(nanobody)具有相同的含义,指克隆抗体重链的可变区,构建仅由一个重链可变区组成的单域抗体(VHH),它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后,再克隆抗体重链的可变区,构建仅由一个重链可变区组成的单域抗体(VHH)。
如本文所用,术语“可变”表示抗体中可变区的某些部分在序列上有所不同,它形成了各种特定抗体对其特定抗原的结合和特异性。然而,可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区,它们大致上呈β-折叠构型,由形成连接环的三个CDR相连,在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I,647-669页(1991))。恒定区不直接参与抗体与抗原的结合,但是它们表现出不同的效应功能,例如参与抗体的依赖于抗体的细胞毒性。
如本领域技术人员所知,免疫偶联物及融合表达产物包括:药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的抗PD-L1蛋白抗体或其片段结合的细胞表面标记物或抗原。
如本文所用,术语“重链可变区”与“VH”可互换使用。
如本文所用,术语“可变区”与“互补决定区(complementarity determiningregion,CDR)”可互换使用。
在本发明的一个优选的实施方式中,所述抗体的重链可变区包括包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中,所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中,术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用,都指特异性结合PD-L1蛋白的多肽,例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地,本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物),只要该可变区与本发明抗体的重链可变区相同或至少90%同源性,较佳地至少95%同源性。
一般,抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述,称为可变区域(CDR),将该段间隔成4个框架区域(FR),4个FR的氨基酸序列相对比较保守,不直接参与结合反应。这些CDR形成环状结构,通过其间的FR形成的β折叠在空间结构上相互靠近,重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣,因为它们中至少部分涉及结合抗原。因此,本发明包括那些具有带CDR的抗体重链可变区的分子,只要其CDR与此处鉴定的CDR具有90%以上(较佳地95%以上,最佳地98%以上)的同源性。
本发明不仅包括完整的抗体,还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此,本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用,术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽,而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的,或(ii)在一个或多个氨基酸残基中具有取代基团的多肽,或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物,例如聚乙二醇)融合所形成的多肽,或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列,或与6His标签形成的融合蛋白)。根据本文的教导,这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有PD-L1蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于):一个或多个(通常为1-50个,较佳地1-30个,更佳地1-20个,最佳地1-10个)氨基酸的缺失、插入和/或取代,以及在C末端和/或N末端添加一个或数个(通常为20个以内,较佳地为10个以内,更佳地为5个以内)氨基酸。例如,在本领域中,用性能相近或相似的氨基酸进行取代时,通常不会改变蛋白质的功能。又比如,在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括:同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽,如包含纳米抗体或其片段的融合蛋白。除了几乎全长的多肽外,本发明还包括了本发明纳米抗体的片段。通常,该片段具有本发明抗体的至少约50个连续氨基酸,较佳地至少约50个连续氨基酸,更佳地至少约80个连续氨基酸,最佳地至少约100个连续氨基酸。
在本发明中,“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比,有至多10个,较佳地至多8个,更佳地至多5个,最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括:只编码成熟多肽的编码序列;成熟多肽的编码序列和各种附加编码序列;成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸,也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50%,较佳地至少70%,更佳地至少80%相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中,“严格条件”是指:(1)在较低离子强度和较高温度下的杂交和洗脱,如0.2×SSC,0.1%SDS,60℃;或(2)杂交时加有变性剂,如50%(v/v)甲酰胺,0.1%小牛血清/0.1%Ficoll,42℃等;或(3)仅在两条序列之间的相同性至少在90%以上,更好是95%以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列,尤其是片段长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。此外,还可将重链的编码序列和表达标签(如6His)融合在一起,形成融合蛋白。
一旦获得了有关的序列,就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前,已经可以完全通过化学合成来得到编码本发明蛋白(或其片段,或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外,还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞,以使其能够表达蛋白质。
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。代表性例子有:大肠杆菌,链霉菌属;鼠伤寒沙门氏菌的细菌细胞;真菌细胞如酵母;果蝇S2或Sf9的昆虫细胞;CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时,能吸收DNA的感受态细胞可在指数生长期后收获,用CaCl2法处理,所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要,转化也可用电穿孔的方法进行。当宿主是真核生物,可选用如下的DNA转染方法:磷酸钙共沉淀法,常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养,表达本发明的基因所编码的多肽。根据所用的宿主细胞,培养中所用的培养基可选自各种常规培养基。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。
本发明的抗体可以单独使用,也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于:荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于:1.放射性核素;2.生物毒;3.细胞因子如IL-2等;4.金纳米颗粒/纳米棒;5.病毒颗粒;6.脂质体;7.纳米磁粒;8.前药激活酶(例如,DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL));10.化疗剂(例如,顺铂)或任何形式的纳米颗粒等。
本发明融合蛋白
如本文所述,“本发明融合蛋白”是指既具有本发明第一方面所述的抗PD-L1纳米抗体,又具有免疫调价分子部分的双功能融合蛋白。
在本发明中,提供了一种融合蛋白,所述纳米抗体融合蛋白从N端到C端具有如式I所示的结构:
Z1-Z2-L-Z3 (式I)
式中,
Z1为如本发明第二方面所述的抗PD-L1纳米抗体的VHH链;
Z2为免疫球蛋白的Fc段;
L为接头序列;
Z3为免疫调节分子部分。
优选地,所述的免疫球蛋白可以是IgG1、IgG2、IgG3或IgG4等。
在一个优选的实施方式中,所述的免疫球蛋白是IgG1,并且所述Z2的氨基酸序列如SEQ ID NO:99所示。在其他的实施方式中,所述Z2的氨基酸序列与如SEQ ID NO:99所示的氨基酸序列相同或基本相同。
在本发明中,所述L为柔性氨基酸接头。优选地,L具有选自下组的氨基酸序列:GGGGS、(GGGGS)2、(GGGGS)3、(GGGGS)4、(GGGGS)5,或其组合。
在一个优选的实施方式中,所述L的氨基酸序列如SEQ ID NO:100所示。在其他的实施方式中,所述L的氨基酸序列与如SEQ ID NO:100所示的氨基酸序列相同或基本相同。
在本发明的一个实施方式中,所述免疫调节分子为TGFβRII胞外结构域。优选地,所述Z3的氨基酸序列如SEQ ID NO:101所示。在其他的实施方式中,所述Z3的氨基酸序列与如SEQ ID NO:101所示的氨基酸序列相同或基本相同。
在本发明中,所述的基本相同是至多有50个(较佳地为1-20个,更佳地为1-10个、更佳地1-5个,最佳地为1-3个)氨基酸不相同,其中,所述的不相同包括氨基酸的取代、缺失或添加。
优选地,所述的基本相同是氨基酸序列与相应氨基酸序列的序列同一性至少为70%、至少为75%、至少为80%、至少为85%、至少为86%、至少为87%、至少为88%、至少为89%、至少为90%、至少为91%、至少为92%、至少为93%、至少为94%、至少为95%、至少为96%、至少为97%、至少为98%,或者至少为99%。
在一个较佳的实施方式中,所述纳米抗体融合蛋白的氨基酸序列如SEQ ID NO:102所示。
TGFβ是上皮间质转化(Epithelial-mesenchymal-transition,EMT)的一个关键诱导因子。同时,TGFβ在肿瘤微环境中具有很强的免疫抑制的作用,进而对肿瘤发生发展、转移和耐药有重要的调控作用。
因此,在本发明的一个实施方式中,选取TGFβ受体II作为融合蛋白中的免疫调节分子。本发明的融合蛋白具有双靶标结合亲和力高、特异性强的优势,从而进一步增强抗肿瘤免疫功能。
药物组合物
本发明还提供了一种组合物。优选地,所述的组合物是药物组合物,它含有上述的抗体或其活性片段或其融合蛋白,以及药学上可接受的载体。通常,可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中,其中pH通常约为5-8,较佳地pH约为6-8,尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药,其中包括(但并不限于):瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合PD-L1蛋白分子,因而可用于治疗肿瘤。此外,还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt%,较佳地0.01-90wt%,更佳地0.1-80wt%)的本发明上述的纳米抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于):盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式,例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量,例如每天约10微克/千克体重-约50毫克/千克体重。此外,本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时,是将安全有效量的免疫偶联物施用于哺乳动物,其中该安全有效量通常至少约10微克/千克体重,而且在大多数情况下不超过约50毫克/千克体重,较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然,具体剂量还应考虑给药途径、病人健康状况等因素,这些都是熟练医师技能范围之内的。
标记的纳米抗体
在本发明的一个优选例中,所述纳米抗体带有可检测标记物。更佳地,所述的标记物选自下组:同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中,抗PD-L1的纳米抗体用胶体金标记,得到胶体金标记的纳米抗体。
本发明的抗PD-L1纳米抗体具有很好的特异性,很高的效价。
检测方法
本发明还涉及检测PD-L1蛋白的方法。该方法步骤大致如下:获得细胞和/或组织样本;将样本溶解在介质中;检测在所述溶解的样本中PD-L1蛋白的水平。
在本发明的检测方法中,所使用的样本没有特别限制,代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒,在本发明的一个优选例中,所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测PD-L1水平的检测试剂盒,该试剂盒包括识别PD-L1蛋白的抗体,用于溶解样本的裂解介质,检测所需的通用试剂和缓冲液,如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
如上所述,本发明的纳米抗体有广泛生物应用价值和临床应用价值,其应用涉及到与PD-L1相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对PD-L1的临床诊断和靶向治疗。
本发明的主要优点包括:
1)本发明纳米抗体高特异性针对人的具有正确空间结构的PD-L1蛋白。
2)本发明纳米抗体的亲和力强。
3)本发明纳米抗体的生产简便。
4)在靶向中和肿瘤微环境的TGF-β基础上抑制PD-1/PD-L1通路,可以使T细胞恢复活性,增强免疫应答,更有效地提高抑制肿瘤发生和发展的效果。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。
实施例1:纳米抗体文库的构建
动物免疫
将1mg人PD-Ll抗原(购自AcroBiosystems)与弗氏佐剂等体积混合,免疫2只羊驼(Llama),每周一次,共免疫4次,剌激B细胞表达抗原特异性的纳米抗体。4次免疫结束后,提取50ml有羊驼外周血,采用淋巴细胞分离液分离得到淋巴细胞。采用RNA提取试剂Trizol(购自Invitrogen)提取总RNA。使用cDNA合成试剂盒(购自Invitrogen)反转录获得羊驼总cDNA。
纳米抗体基因扩增
第一轮PCR,从cDNA中扩增出IgG2、IgG3序列:
表1.第一轮PCR引物
名称 | 序列(5’到3’) |
上游引物 | GTCCTGGCTGCTCTTCTACAAGG |
下游引物 | GGTACGTGCTGTTGAACTGTTCC |
第一轮PCR产物进行琼脂糖凝胶电泳,切胶回收750bp处的片段用于第二轮VHH序列扩增。第二轮PCR扩增引物如下:
表2.第二轮PCR引物
名称 | 序列(5’到3’) |
上游引物 | CTAGTGCGGCCGCcTGGAGACGGTGACCTGGGT |
下游引物 | CGCGGATCCCAGGTGCAGCTGCAGGAGTCTGGRGGAGG |
以第二轮PCR产物为模板,进行第三轮PCR,为VHH基因加上同源臂,第三轮PCR扩增引物如下:
表3.第三轮PCR引物
利用PCR纯化试剂盒(购自QIAGEN)回收目的片段。
文库构建
将线性化的酵母展示载体和第三轮的PCR产物混合后电转化入酿酒酵母(购自ATCC)中,构建来自两个动物的抗PD-L1纳米抗体文库并测定库容,库容大小分别为4.47×107和4.14×107。
实施例2:PD-L1纳米抗体的筛选
人PD-L1蛋白的生物素化标记
取适量体积的双蒸水溶解人PD-L1蛋白(购自AcroBiosystems),按照生物素标记试剂盒(购自Thermo)产品说明书,将生物素溶解后与蛋白溶液混合,于4℃孵育2小时。用脱盐柱(购自Thermo)去除多余的生物素,脱盐柱预处理及样品收集操作均参考产品说明书步骤进行。
MACS富集能与PD-L1特异性结合的酵母
将实施例2中构建的VHH文库接种于SD-CAA扩增培养基(1L SD-CAA扩增培养基中含6.7g YNB、5g酪蛋白氨基酸、13.62g Na2HPO4·12H2O、7.44g NaH2PO4和2%葡萄糖)中,接种的酵母细胞数>10×文库容量(起始扩增浓度=0.5OD600/ml),30℃,225rpm培养过夜。取10×库容量的酵母细胞,3000rpm×5min离心(以下离心操作均同此)去除培养基,用SD-CAA诱导培养基重悬酵母细胞,调整起始浓度为0.5OD600/ml,诱导过夜。测定诱导后的文库浓度,取10×库容量的酵母细胞,离心去除培养基。用50ml洗涤缓冲液(PBS+0.5%BSA+2mMEDTA)重悬酵母细胞,离心去除上清。用10ml洗涤缓冲液重悬酵母细胞。
加入生物素标记的PD-L1蛋白(终浓度100mM),室温孵育30min,离心收集酵母细胞,并用50ml洗涤缓冲液洗涤酵母3遍。用5ml洗涤缓冲液重悬酵母细胞,并加入200μl SA磁珠(购自美天旎),颠倒孵育10min。用洗涤缓冲液洗涤酵母和磁珠混合物3遍,将混合物加入LS柱(购自美天旎)中。将LS柱放在磁力架上,用洗涤缓冲液洗涤去除非特异性结合的酵母细胞。将柱子从磁力架上取出,加入洗涤缓冲液洗脱酵母。洗脱下来的酵母离心后转入200ml SD-CAA扩增培养基中进行扩增。
流式细胞分选获得高亲和力酵母细胞
将经过MACS富集的酵母细胞接种于SD-CAA扩增培养基中,起始扩增浓度=0.5OD600/ml。30℃,225rpm摇瓶培养过夜。用SD-CAA诱导培养基(1L SD-CAA诱导培养基中含6.7g YNB,5g酪蛋白氨基酸,13.62g Na2HPO4·12H2O,7.44g NaH2PO4及2%半乳糖,2%棉子糖和0.1%葡萄糖)重悬酵母细胞,起始浓度为0.5OD600/ml,诱导过夜。加入1:200稀释的anti-c-Myc鼠源抗体(购自Thermo)和100nM生物素标记的PD-L1抗原,室温孵育10min。加入PBS清洗酵母3遍,加入1:500稀释的羊抗鼠IgG(H+L)Alexa Fluor Plus 488(购自Invitrogen)和链霉亲和素APC偶联荧光抗体(购自Invitrogen),4℃避光孵育15min。加入2ml PBS重悬细胞,使用BD FACSAraiII仪器进行分选获得可与PD-L1抗原有较高结合能力的酵母。
PD-L1纳米抗体候选分子抗体基因的调取
通过MACS和FACS富集得到的能与PD-L1抗原有较高结合能力的酵母菌液,在SD-CAA扩增培养基中30℃,225rpm培养过夜,按照酵母质粒抽提试剂盒(购自天根)操作抽提酵母质粒。质粒通过电转化入Top10感受态细胞(购自天根),涂布氨苄抗性平板,于37℃培养过夜。挑取单克隆测序获得VHH基因序列。
实施例3:重链抗体的构建及表达纯化
抗体基因构建入pCDNA3.1表达载体
将VHH基因序列和人IgG1(LALA突变)Fc段相连,利用同源重组酶(购自Vazyme)和EcoR I/Not I双酶切线性化的pCDNA3.1载体中,流程按照商品说明书。同源重组产物化转入Top10感受态细胞,涂布氨苄抗性平板,37℃培养过夜,挑取单克隆测序。
细胞转染
采用ExpiCHOTM表达系统试剂盒(购自Thermo),将质粒转入Expi-CHO细胞中,转染方法按照商品说明书,细胞培养5天后收集上清利用蛋白A磁珠(购自金斯瑞)分选法纯化目的蛋白。将磁珠用适当体积的结合缓冲液(PBS+0.1%吐温20,pH 7.4)重悬(1-4倍磁珠体积)后加入至待纯化样品中,室温孵育1小时,期间温柔振荡。样品置于磁力架上(购自海狸),弃去上清,磁珠用结合缓冲液清洗3遍。按照磁珠体积的3-5倍体积加入洗脱缓冲液(0.1M sodium citrate,pH 3.2)室温振荡5-10min,置回磁力架上,收集洗脱缓冲液,转移至已加入中和缓冲液(1M Tris,pH 8.54)的收集管中混匀。
实施例4:纯化抗PD-L1抗体与人PD-L1结合
通过转染克隆到MCS的人PD-L1 cDNA(购自Sino Biological)的pCHO1.0载体(购自Invitrogen)产生过表达人PD-L1的CHO细胞(CHO-hPD-L1细胞)。将扩大培养的CHO-hPD-L1细胞调整细胞密度至2×106细胞/ml,100μl/孔加入96孔流式板,离心备用。将纯化的PD-L1抗体用PBS稀释,400nM开始3倍稀释共12个点,将上述稀释好的样品100μl/孔加入上述带有细胞的96孔流式板中,4℃孵育30min,PBS清洗两次。100μl/孔加入用PBS稀释100倍的羊F(ab’)2抗人IgG-Fc(PE)(购自Abcam),4℃孵育30min,PBS清洗两次。100μl/孔加入PBS重悬细胞,在CytoFlex(Bechman)流式细胞仪上进行检测并计算对应的MFI。
在如上方法的测定实验中,实验结果如图1所示,本发明所有的纯化样品和CHO-hPD-L1细胞均有结合活性,且部分纯化样品的结合活性与对照抗体ATE(US20130034559)相似。
实施例5:PD-L1抗体亲和力测定
ForteBio亲和力测定按照现有的方法(Estep,P等人,基于溶液的高通量抗体-抗原亲和力和表位分级的测量,MAbs,2013.5(2):p.270-8)进行。简言之,传感器在分析缓冲液中线下平衡30min,然后线上检测60s建立基线,在线加载如上所述获得的经纯化的抗体至AHQ传感器上。再将传感器放入100nM的PD-L1抗原中作用5min,之后将传感器转移至PBS中解离5min。使用1:1结合模型进行动力学的分析。
表4.候选分子亲和力
编号 | KD(M) | Kon(1/Ms) | Koff(1/s) |
C-Ye-02 | 7.98E-08 | 4.69E+04 | 3.75E-03 |
C-Ye-04 | 4.89E-09 | 1.03E+05 | 5.05E-04 |
C-Ye-06 | 1.34E-07 | 4.35E+04 | 5.81E-03 |
C-Ye-17 | 3.96E-07 | 2.58E+04 | 1.02E-02 |
C-Ye-18 | 5.39E-09 | 8.33E+04 | 4.49E-04 |
C-Ye-20 | 4.86E-08 | 9.06E+04 | 4.40E-03 |
C-Ye-24 | 1.95E-08 | 7.71E+04 | 1.51E-03 |
C-Ye-26 | 3.41E-08 | 9.41E+04 | 3.21E-03 |
C-Ye-27 | 5.79E-08 | 5.45E+04 | 3.15E-03 |
C-Ye-30 | 3.94E-08 | 5.32E+04 | 2.10E-03 |
C-Ye-32 | 4.43E-08 | 5.83E+04 | 2.58E-03 |
C-Ye-34 | 2.44E-08 | 7.81E+04 | 1.91E-03 |
C-Ye-39 | 5.03E-07 | 2.12E+04 | 1.07E-02 |
C-Ye-42 | 3.89E-08 | 5.70E+04 | 2.22E-03 |
实施例6:PD-L1抗体基因改造
为去除C-Ye-18中的潜在糖基化位点,将C-Ye-18氨基酸序列中CDRH2部分点突变成表5中的6种形式:
表5.C-Ye-18 CDR区突变序列
编号 | CDRH2区 |
C-Ye-18 | SINSSSSSTYYRDSVKG |
C-Ye-18-1 | SINSGSSSTYYRDSVKG |
C-Ye-18-2 | SISSSSSSTYYRDSVKG |
C-Ye-18-3 | SIGSSSSSTYYRDSVKG |
C-Ye-18-4 | SIYSGSSSTYYRDSVKG |
C-Ye-18-5 | SINNSDSSTYYRDSVKG |
C-Ye-18-6 | SINSGSSSTYYRDSVKG |
本研究利用IMGT(http://www.imgt.org)对C-Ye-18 CDR区突变序列的人源化水进行了评估,结果如表6所示,所有C-Ye-18的突变型人源化水平均高于87%,符合后期药物开发要求。
表6.C-Ye-18 CDR区突变序列和人的同源性
编号 | Germline | 同源性 |
C-Ye-18 | IGHV3-74*01 | 87.80% |
C-Ye-18-1 | IGHV3-74*01 | 88.80% |
C-Ye-18-2 | IGHV3-74*01 | 87.80% |
C-Ye-18-3 | IGHV3-74*01 | 87.80% |
C-Ye-18-4 | IGHV3-74*01 | 87.80% |
C-Ye-18-5 | IGHV3-74*01 | 87.80% |
C-Ye-18-6 | IGHV3-74*01 | 87.80% |
蛋白构建及表达纯化方法同实施例3,利用HPLC检测获得蛋白的纯度。HPLC方法如下,流动相:150mM Na2HPO4·12H2O,pH7.0。色谱条件:检测波长:280nm,柱温:25℃,流速:0.35ml/min,检测时间:20min,Zenix-C SEC-300色谱柱(SEPAX 4.6×300mm,3μm)。
表7.C-Ye-10突变抗体的纯度检测结果
编号 | 单体比例(%) |
C-Ye-18 | 90.70 |
C-Ye-18-1 | 97.40 |
C-Ye-18-2 | 96.40 |
C-Ye-18-3 | 98.50 |
C-Ye-18-4 | 73.70 |
C-Ye-18-5 | 83.00 |
C-Ye-18-6 | 95.80 |
实施例7:C-Ye-18突变样品与人PD-L1结合
本实验检测纯化获得的C-Ye-18突变样品与CHO-hPD-L1细胞结合活性,实验方法同实施例4,实验结果如图2所示,C-Ye-18突变样品与CHO-hPD-L1细胞有很好的结合活性,水平与C-Ye-18以及对照抗体ATE相当。
实施例8:C-Ye-18突变样品亲和力测定
本实验检测纯化获得的C-Ye-18突变样品与人PD-L1结合亲和力,实验方法同实施例5,实验结果如表8所示,C-Ye-18突变样品与人PD-L1蛋白有很好的结合活性。
表8 C-Ye-18突变样品亲和力
编号 | KD(M) | kon(1/Ms) | kdis(1/s) |
C-Ye-18-1 | 4.13E-09 | 2.46E+05 | 1.02E-03 |
C-Ye-18-2 | 5.41E-09 | 2.34E+05 | 1.27E-03 |
C-Ye-18-3 | 7.03E-09 | 2.41E+05 | 1.70E-03 |
C-Ye-18-4 | 6.40E-09 | 2.41E+05 | 1.54E-03 |
C-Ye-18-5 | 4.08E-09 | 2.72E+05 | 1.11E-03 |
C-Ye-18-6 | 6.00E-09 | 2.32E+05 | 1.39E-03 |
实施例9:C-Ye-18突变样品阻断PD-L1与PD-1结合
通过转染克隆到MCS的人PD-1 cDNA(购自Sino Biological)的pCHO1.0载体(购自Invitrogen)产生过表达人PD-1的CHO细胞(CHO-hPD-1细胞)。将扩大培养的CHO-hPD-1细胞调整细胞密度至2×106cells/ml,100μl/孔加入96孔流式板,离心备用。将纯化的突变样品用PBS稀释,400nM开始3倍稀释共12个点,将上述稀释好的样品60μl/孔加入96孔样品稀释板,同时60μl/孔加入Biotinylated human PD-L1蛋白(购自AcroBiosystems),终浓度为500ng/ml,与突变样品4℃孵育30min。将共孵育样品100μl/孔加入上述带有细胞的96孔流式板中,4℃孵育30min,PBS清洗两次。100μl/孔加入用PBS稀释100倍的链霉素R-藻红蛋白缀合物(购自Thermo Fisher),4℃孵育30min,PBS清洗两次。100μl/孔加入PBS重悬细胞,在CytoFlex(Bechman)流式细胞仪上进行检测并计算对应的MFI。
在如上方法的测定实验中,实验结果如图3所示,本发明所有的突变样品均可以阻断PD-L1与PD-1的结合,且阻断水平与C-Ye-18以及对照抗体ATE相当。
实施例10:C-Ye-18突变样品的热稳定性
利用DSC(Differential scanning calorimetry,差示扫描量热法)检测不同抗体的热稳定性。将样品浓缩后用PBS稀释到1mg/ml;将5000×荧光显色剂Cypro Orange(购于Bio-Rad)用超纯水稀释50倍得到100×荧光显色剂Sypro Orange。取50μl 1mg/ml的样品加入10μl 100×荧光显色剂Sypro Orange、40μl超纯水,混匀后,取30μl加入到96孔PCR板中,每个样品做3个复孔,放入PCR仪中,设置升温程序为:25℃恒温5min,以0.5℃/min的速度升温至99℃。程序结束后在“Melt Curve”图中读取曲线的最低点的温度值,即为样品的Tm值。具体结果如下表9所示
表9 C-Ye-18突变抗体的Tm值
实施例11:混合淋巴细胞反应实验
本实施例通过混合淋巴细胞反应实验(MLR)检测C-Ye-18突变样品激活T细胞的活性。具体实验方法如下。
复苏PBMC细胞(购自SAILY BIO,SLB-HPB),离心,用10ml X-VIVO-15培养基(购自LONZA)重悬PBMC,于细胞培养箱内37℃贴壁培养2h,吸去未贴壁细胞。加入10ml DC培养基:X-VIVO-15培养基加入10ng/ml GM-CSF(购自R&D),20ng/ml IL-4,培养3天,补加5ml DC培养基,继续培养至第6天,加入DC成熟培养基:X-VIVO-15培养基加入1000U/ml TNF-α(购自R&D),10ng/ml IL-6(购自R&D),5ng/ml IL-1β(购自R&D),1μM PGE2(购自Tocris),培养2天,收集成熟的DC细胞,用X-VIVO-15培养基调整细胞密度为2×105细胞/ml。
复苏另一位捐献者的PBMC细胞(购自SAILY BIO,SLB-HPB),离心,用10ml X-VIVO-15培养基重悬PBMC。用CD4+T细胞分选试剂盒(购自Stemcell)富集CD4+T细胞,X-VIVO-15重悬CD4+T细胞,调整细胞密度为2×106cells/ml.将CD4+T细胞与上述收集的成熟DC细胞按1:1比例混合,100μl/孔加入96孔U底板。
用X-VIVO-15培养基稀释C-Ye-18突变样品,200nM开始3倍稀释共9个点,100μl/孔加入上述混合细胞孔,培养5天,收集上清,ELISA(购自eBioscience)方法检测IFN-γ和IL2表达量。
结果如图4所示,C-Ye-18突变样品,C-Ye-18-1、C-Ye-18-5、C-Ye-18-6均在MLR实验中显示较好的生物学活性,且激活水平与对照抗体ATE相似或优于对照抗体。
实施例12融合蛋白PD-Ll/TGF-b trap克隆和表达
在本实施例中,采用TGFβRII胞外结构域(SEQ ID NO:101)作为融合蛋白中免疫调节分子部分,将PD-L1抗体作为融合蛋白的靶向部分,形成PD-L1抗体/TGFβRII胞外区融合蛋白(PM8001)。
利用分子克隆技术将本发明PD-L1单链抗体的C末端氨基酸通过(G4S)4G连接TGFβRII胞外区,通过Expi-CHO表达系统进行常规表达,表达纯化方法同实施例3,得到结构如图5所示的融合蛋白PM8001。
实施例13 PM8001分子与人PD-L1结合
检测纯化的PD-L1抗体(C-Ye-18-5)、PM8001分子、TGF-βR2-Fc融合蛋白、阳性对照M7824(WO2015/118175A2)及阴性对照IgG蛋白和细胞表面PD-L1结合活性的方法同实施例4。在如上方法的测定实验中,实验结果如图6所示,本发明PM8001分子和CHO-hPD-L1细胞有结合活性,且结合活性与阳性对照分子M7824相似。
实施例14 PM8001分子阻断PD-L1蛋白与PD-1细胞结合
检测纯化的PD-L1抗体(C-Ye-18-5)、PM8001分子、TGF-βR2-Fc融合蛋白、阳性对照M7824及阴性对照IgG蛋白阻断PD-L1蛋白和PD-1细胞结合活性的方法同实施例9。在如上方法的测定实验中,实验结果如图7所示,本发明PM8001分子可以阻断PD-L1蛋白与PD-1细胞的结合,且阻断水平与阳性对照分子M7824相当。
实施例15 PM8001分子与人TGF-β家族蛋白ELISA水平结合实验
用ELISA包被液将人TGF-β1(acrobiosystems,TG1-H421),TGF-β2(PeproTech,100-35B),TGF-β3(PeproTech,100-36E)蛋白稀释后加入ELISA板,4℃包被过夜。弃去包被液,250μl/孔加入PBST洗3次,用5%BSA室温封闭1小时备用。将纯化的PD-L1抗体(C-Ye-18-5),PM8001分子,TGF-βR2-Fc融合蛋白,阳性对照M7824用梯度稀释后加入封闭好的ELISA板,室温孵育2小时。加入PBST洗3次,向孔中加入羊抗人Fc-HRP(abcam,ab97225),室温孵育1小时,加入PBST洗3次后加入ELISA显色液,室温放置3min后加入ELISA终止液,读取450nm处吸光度数值。
在如上方法的测定实验中,实验结果如图8所示,本发明PM8001分子在ELISA水平与人TGF-β1,TGF-β3蛋白有较好的结合,与人TGF-β2蛋白结合活性较弱,且结合水平与阳性对照分子M7824相当。
实施例16 PM8001分子阻断TGF-β/SMAD信号通路实验
取适量293-TGF-β/SMAD效应胞接种于96孔细胞培养白底板,置于37℃,5%CO2培养箱培养过夜。将纯化的PD-L1抗体(C-Ye-18-5),PM8001分子,TGF-βR2-Fc融合蛋白,阳性对照M7824梯度稀释后和TGF-β1(acrobiosystems,TG1-H421)混合,室温孵育30min。将上述混合物加入带有细胞的白底板中继续培养过夜。向每孔中加入Bio-Glo TM试剂(Promega),使用多功能酶标仪读取荧光信号值。
在如上方法的测定实验中,实验结果如图9所示,本发明PM8001分子可以体外阻断TGF-β/SMAD信号通路,且阻断水平与阳性对照分子M7824相当。
实施例17混合淋巴细胞反应实验
混合淋巴细胞检测纯化的PD-L1抗体(C-Ye-18-5)、PM8001分子、TGF-βR2-Fc融合蛋白、阳性对照M7824及阴性对照IgG蛋白激活人T淋巴细胞的方法同实施例9。结果如图10所示,本发明PM8001分子在MLR实验中显示较好的生物学活性,且激活水平相当或优于阳性对照分子M7824。
实施例18 PM8001大鼠药代动力学评价
实验用SD大鼠6只,雌雄各半,12/12小时光/暗调节,温度24±2℃,湿度40-70%,自由进水饮食。购自浙江维通利华实验技术有限公司。实验当天对SD大鼠单次尾静脉注射PM8001分子,注射剂量为10mg/Kg。
取血时间点:给药后3min、4小时、10小时、24小时、48小时、72小时、120小时、168小时、240小时、336小时、504小时、672小时于大鼠颈静脉采血。全血样品2-8℃放置30分钟,12000rpm离心5min收集血清,所得血清再于2-8℃,12000rpm离心5min,-80℃保存,ELISA检测血清中游离PM8001分子量。结果如表10所示,本发明PM8001游离状态分子在SD大鼠体内半衰期约为146小时。
表10 PM8001在SD大鼠中的T1/2
实施例19 PM8001的肿瘤抑制活性研究
本实验采用表达人PD-L1的MC38细胞(h-PD-L1 KI MC38)在PD-L1转基因小鼠测定PM8001的抗肿瘤作用。首先采用皮下接种的方式建立h-PD-L1 KI MC38荷瘤小鼠模型,待平均肿瘤体积长至80-120mm3时进行分组,单次腹腔注射给予不同抗体和不同剂量的治疗,监测各组小鼠瘤体积和体重变化,监测频率均为2次/周,连续监测3周,给药剂量和方式如表11,小鼠瘤体积变化如图11所示。
表11 PM8001的肿瘤抑制活性实验方案
实验结果如图11所示。接种了h-PD-L1 KI MC38后阴性对照组肿瘤体积不断增大,TGF-βR II-Fc和C-Ye-18-5单用组肿瘤生长均受到抑制,而PM8001组相比于TGF-βR II-Fc和C-Ye-18-5组得到了更好的控制,说明PM8001有明显的肿瘤抑制作用,与阳性对照组相当甚至稍优。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
本发明的序列信息
SEQ ID NO:1 C-Ye-18 CDR1氨基酸序列
GFTFSSYWMY
SEQ ID NO:2 C-Ye-18 CDR2氨基酸序列
SINSSSSSTYYRDSVKG
SEQ ID NO:3 C-Ye-18 CDR3氨基酸序列
AKDPGGYA
SEQ ID NO:4C-Ye-18 VHH氨基酸序列
EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS
SEQ ID NO:5 C-Ye-18 VHH核苷酸序列
GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTAGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:6 C-Ye-04 CDR1氨基酸序列
SGFTFSSYWMY
SEQ ID NO:7 C-Ye-04 CDR2氨基酸序列
SINTSSSSTYYRDSVKG
SEQ ID NO:8 C-Ye-04 CDR3氨基酸序列
AKDPGGYA
SEQ ID NO:9 C-Ye-04 VHH氨基酸序列
QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINTSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS
SEQ ID NO:10 C-Ye-04 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATACTAGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:11 C-Ye-02 CDR1氨基酸序列
GRTFNNSAMGAMG
SEQ ID NO:12 C-Ye-02 CDR2氨基酸序列
TITWSSGSSFYANSVKG
SEQ ID NO:13 C-Ye-02 CDR3氨基酸序列
ASRKLGGVVTVVTSYDF
SEQ ID NO:14 C-Ye-02 VHH氨基酸序列
QVQLQESGGGLVQAGGSLRLSCAASGRTFNNSAMGAMGWFRQAPGKEREFVATITWSSGSSFYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASRKLGGVVTVVTSYDFWGQGTQVTVSS
SEQ ID NO:15C-Ye-02 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAATAACTCGGCCATGGGCGCCATGGGATGGTTCCGCCAGGCGCCAGGGAAAGAGCGTGAGTTTGTCGCGACAATTACCTGGAGTAGTGGTAGCTCATTTTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCATCACGCAAATTGGGAGGGGTTGTAACGGTAGTTACTTCGTATGACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:16 C-Ye-06 CDR1氨基酸序列
GRTFDNYAMGAMG
SEQ ID NO:17 C-Ye-06 CDR2氨基酸序列
TITWSSGSSFYANSVKG
SEQ ID NO:18 C-Ye-06 CDR3氨基酸序列
ASRKLGGVVTVVTSYDF
SEQ ID NO:19 C-Ye-06 VHH氨基酸序列
QVQLQESGGGLVQPGGSLRLSCAASGRTFDNYAMGAMGWFRQAPGKEREFVATITWSSGSSFYANSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCASRKLGGVVTVVTSYDFWGQGTQVTVSS
SEQ ID NO:20 C-Ye-06 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCGATAACTATGCCATGGGCGCCATGGGATGGTTCCGCCAGGCGCCAGGGAAAGAGCGTGAGTTTGTCGCGACAATTACCTGGAGTAGTGGTAGCTCATTTTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGACGACACGGCCGTTTATTACTGTGCATCACGCAAATTGGGAGGGGTTGTAACGGTAGTTACTTCGTATGACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:21 C-Ye-09 CDR1氨基酸序列
GRTFSTYAVG
SEQ ID NO:22 C-Ye-09 CDR2氨基酸序列
GRLTWSGSRTYYADSVKG
SEQ ID NO:23 C-Ye-09 CDR3氨基酸序列
AADYRSNSTWSLQSPARYEN
SEQ ID NO:24 C-Ye-09 VHH氨基酸序列
QVQLQESGGGLVQAGDSLGLSCTASGRTFSTYAVGWFRQAPGKGREFVGRLTWSGSRTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAADYRSNSTWSLQSPARYENWGQGTQVTVSS
SEQ ID NO:25 C-Ye-09 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGGGACTCTCCTGTACAGCCTCTGGACGCACCTTCAGTACCTATGCCGTGGGGTGGTTCCGCCAGGCTCCAGGGAAGGGGCGTGAATTTGTAGGACGTCTTACATGGAGCGGGAGTAGAACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCCGACTACCGAAGTAACAGTACCTGGTCCCTGCAAAGCCCGGCACGTTATGAAAATTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:26 C-Ye-10 CDR1氨基酸序列
GRTVSNYAMG
SEQ ID NO:27 C-Ye-10 CDR2氨基酸序列
RITGSGSSTFYADSVKG
SEQ ID NO:28 C-Ye-10 CDR3氨基酸序列
AADRWRSMVTRSDPREYEN
SEQ ID NO:29 C-Ye-10 VHH氨基酸序列
QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS
SEQ ID NO:30 C-Ye-10 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:31 C-Ye-17 CDR1氨基酸序列
GRTVSNYAMG
SEQ ID NO:32 C-Ye-17 CDR2氨基酸序列
RITGSGSSTFYADSVKG
SEQ ID NO:33 C-Ye-17 CDR3氨基酸序列
AADRWRSMVTRSDPREYEN
SEQ ID NO:34 C-Ye-17 VHH氨基酸序列
QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGLFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS
SEQ ID NO:35 C-Ye-17 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCTATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:36 C-Ye-20 CDR1氨基酸序列
GRTVSNYAMG
SEQ ID NO:37 C-Ye-20 CDR2氨基酸序列
RITGSGSSTFYADSVKG
SEQ ID NO:38 C-Ye-20 CDR3氨基酸序列
AADRWRSMVTRSYPREYEN
SEQ ID NO:39 C-Ye-20 VHH氨基酸序列
QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCAADRWRSMVTRSYPREYENWGQGTQVTVSS
SEQ ID NO:40 C-Ye-20 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACGCGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTTACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:41 C-Ye-24 CDR1氨基酸序列
GRTVSNYAMG
SEQ ID NO:42 C-Ye-24 CDR2氨基酸序列
RITGSGRTTYYADSVKG
SEQ ID NO:43 C-Ye-24 CDR3氨基酸序列
AADRWRSMVTRSDPREYEN
SEQ ID NO:44 C-Ye-24 VHH氨基酸序列
QVQLQESGGGVVQAGDSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGRTTYYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS
SEQ ID NO:45 C-Ye-24 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGTGTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTCGTACCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:46 C-Ye-26 CDR1氨基酸序列
GRTVSNYAMG
SEQ ID NO:47 C-Ye-26 CDR2氨基酸序列
RITGSGSSTFYADSVKG
SEQ ID NO:48 C-Ye-26 CDR3氨基酸序列
AADRWRSMVTRSDPRDYEN
SEQ ID NO:49 C-Ye-26 VHH氨基酸序列
QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPRDYENWGQGTQVTVSS
SEQ ID NO:50 C-Ye-26 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGATTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:51 C-Ye-27 CDR1氨基酸序列
GRTFSRYAVG
SEQ ID NO:52 C-Ye-27 CDR2氨基酸序列
AITWSGGYTYYADSVKG
SEQ ID NO:53 C-Ye-27 CDR3氨基酸序列
AVDTRNVIGPRAGDY
SEQ ID NO:54 C-Ye-27 VHH氨基酸序列
QVQLQESGGGLVQAGGSLRLSCAASGRTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS
SEQ ID NO:55 C-Ye-27 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:56 C-Ye-30 CDR1氨基酸序列
GSTFSRYAVG
SEQ ID NO:57 C-Ye-30 CDR2氨基酸序列
AITWSGGYTYYADSVKG
SEQ ID NO:58C-Ye-30 CDR3氨基酸序列
AVDTRNVIGPRAGDY
SEQ ID NO:59 C-Ye-30 VHH氨基酸序列
QVQLQESGGGLVQAGGSLRLSCAASGSTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS
SEQ ID NO:60 C-Ye-30 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:61 C-Ye-32 CDR1氨基酸序列
GRTFSRYAVG
SEQ ID NO:62 C-Ye-32 CDR2氨基酸序列
AITWSGGYTYYADSVKG
SEQ ID NO:63 C-Ye-32 CDR3氨基酸序列
AVDTRNVIGPRAGDY
SEQ ID NO:64 C-Ye-32 VHH氨基酸序列
QVQLQESGGGLVQAGGSLRLSCAASGRTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTIYLQMNSLNVEDTGVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS
SEQ ID NO:65 C-Ye-32 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGATCTATCTCCAAATGAACAGCCTGAACGTTGAGGACACGGGCGTTTATTACTGCGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:66 C-Ye-34 CDR1氨基酸序列
AASGRTFSRFAMG
SEQ ID NO:67 C-Ye-34 CDR2氨基酸序列
AISWSGGMIYYTDSVKG
SEQ ID NO:68 C-Ye-34 CDR3氨基酸序列
AVDTRNVIGPRAGDY
SEQ ID NO:69 C-Ye-34 VHH氨基酸序列
QVQLQESGGGLVQAGGSLRLSCAASGRTFSRFAMGWFRQAPGKEREFVAAISWSGGMIYYTDSVKGRFTISRDNAKNMLYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS
SEQ ID NO:70 C-Ye-34 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACTTTCAGTAGGTTTGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCCGCTATTAGCTGGAGTGGTGGTATGATATACTATACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACATGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:71 C-Ye-39 CDR1氨基酸序列
GRAFSVYPMA
SEQ ID NO:72 C-Ye-39 CDR2氨基酸序列
RLTYTSNTFYADSVKG
SEQ ID NO:73 C-Ye-39 CDR3氨基酸序列
AVENRSSSWSLQSPARYDD
SEQ ID NO:74 C-Ye-39 VHH氨基酸序列
QVQLQESGGGLVQAGGSLRLSCTASGRAFSVYPMAWFRQAPGKEREFIARLTYTSNTFYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVENRSSSWSLQSPARYDDWGQGTQVTVSS
SEQ ID NO:75 C-Ye-39 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCATGTACAGCCTCTGGACGCGCCTTCAGTGTCTACCCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTATAGCACGTCTTACGTATACTAGTAACACATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAGATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGGTCGAGAACCGCAGTAGTAGTTGGTCCCTGCAAAGCCCGGCACGTTATGATGACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:76 C-Ye-42 CDR1氨基酸序列
GRTGSRYAVG
SEQ ID NO:77 C-Ye-42 CDR2氨基酸序列
AITWSGGYTYYADSVKG
SEQ ID NO:78 C-Ye-42 CDR3氨基酸序列
AVDTRNVIGPRAGDY
SEQ ID NO:79 C-Ye-42 VHH氨基酸序列
QVQLQESGGGLVQAGGSLRLSCAASGRTGSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS
SEQ ID NO:80 C-Ye-42 VHH核苷酸序列
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCGGCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGATGTATCTGCAAATGAACAGCCTAAAACCTGAAGACACGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:81 C-Ye-18-1 CDR2氨基酸序列
SINSGSSSTYYRDSVKG
SEQ ID NO:82 C-Ye-18-1 VHH氨基酸序列
EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSGSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS
SEQ ID NO:83 C-Ye-18-1 VHH核苷酸序列
GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTGGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:84 C-Ye-18-2 CDR2氨基酸序列
SISSSSSSTYYRDSVKG
SEQ ID NO:85 C-Ye-18-2 VHH氨基酸序列
EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSISSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS
SEQ ID NO:86 C-Ye-18-2 VHH核苷酸序列
GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAGTAGTAGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:87 C-Ye-18-3 CDR2氨基酸序列
SIGSSSSSTYYRDSVKG
SEQ ID NO:88 C-Ye-18-3 VHH氨基酸序列
EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSIGSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS
SEQ ID NO:89 C-Ye-18-3 VHH核苷酸序列
GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTGGTAGTAGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:90 C-Ye-18-4 CDR2氨基酸序列
SIYSGSSSTYYRDSVKG
SEQ ID NO:91 C-Ye-18-4 VHH氨基酸序列
EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSIYSGSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS
SEQ ID NO:92 C-Ye-18-4 VHH核苷酸序列
GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTTACAGTGGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:93 C-Ye-18-5 CDR2氨基酸序列
SINNSDSSTYYRDSVKG
SEQ ID NO:94 C-Ye-18-5 VHH氨基酸序列
EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSDSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS
SEQ ID NO:95 C-Ye-18-5 VHH核苷酸序列
GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTGACAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:96 C-Ye-18-6 CDR2氨基酸序列
SINSGSSSTYYRDSVKG
SEQ ID NO:97 C-Ye-18-6 VHH氨基酸序列
EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSISGSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS
SEQ ID NO:98 C-Ye-18-6 VHH核苷酸序列
GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAGTGGTAGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT
SEQ ID NO:99 IgG1 Fc片段氨基酸序列
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO:100融合蛋白linker氨基酸序列
GGGGSGGGGSGGGGSGGGGSG
SEQ ID NO:101 TGFβRII胞外结构域氨基酸序列
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
SEQ ID NO:102 PM8001氨基酸序列
EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSDSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
序列表
<110> 普米斯生物技术(珠海)有限公司
<120> 一种抗PD-L1纳米抗体及其衍生物和用途
<130> P2019-0559
<160> 102
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 1
Gly Phe Thr Phe Ser Ser Tyr Trp Met Tyr
1 5 10
<210> 2
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 2
Ser Ile Asn Ser Ser Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 3
<211> 8
<212> PRT
<213> 人工序列(artificial sequence)
<400> 3
Ala Lys Asp Pro Gly Gly Tyr Ala
1 5
<210> 4
<211> 115
<212> PRT
<213> 人工序列(artificial sequence)
<400> 4
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Asn Ser Ser Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 5
<211> 345
<212> DNA
<213> 人工序列(artificial sequence)
<400> 5
gaggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgcgactc 60
tcctgtgcag cctctggatt caccttcagt agctactgga tgtattggct ccgtcaggct 120
ccagggaagg ggctcgagtg ggtctcatct attaatagta gtagtagtag cacatactat 180
cgagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctgcaaatga acagtctgaa atctgaggac acggccgtgt attactgtgc aaaagatcct 300
ggtgggtacg ccaaaggcca ggggacccag gtcaccgtct ccagt 345
<210> 6
<211> 11
<212> PRT
<213> 人工序列(artificial sequence)
<400> 6
Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met Tyr
1 5 10
<210> 7
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 7
Ser Ile Asn Thr Ser Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 8
<211> 8
<212> PRT
<213> 人工序列(artificial sequence)
<400> 8
Ala Lys Asp Pro Gly Gly Tyr Ala
1 5
<210> 9
<211> 115
<212> PRT
<213> 人工序列(artificial sequence)
<400> 9
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Asn Thr Ser Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 10
<211> 345
<212> DNA
<213> 人工序列(artificial sequence)
<400> 10
caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgcgactc 60
tcctgtgcag cctctggatt caccttcagt agctactgga tgtattggct ccgtcaggct 120
ccagggaagg ggctcgagtg ggtctcatct attaatacta gtagtagtag cacatactat 180
cgagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctgcaaatga acagtctgaa atctgaggac acggccgtgt attactgtgc aaaagatcct 300
ggtgggtacg ccaaaggcca ggggacccag gtcaccgtct ccagt 345
<210> 11
<211> 13
<212> PRT
<213> 人工序列(artificial sequence)
<400> 11
Gly Arg Thr Phe Asn Asn Ser Ala Met Gly Ala Met Gly
1 5 10
<210> 12
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 12
Thr Ile Thr Trp Ser Ser Gly Ser Ser Phe Tyr Ala Asn Ser Val Lys
1 5 10 15
Gly
<210> 13
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 13
Ala Ser Arg Lys Leu Gly Gly Val Val Thr Val Val Thr Ser Tyr Asp
1 5 10 15
Phe
<210> 14
<211> 127
<212> PRT
<213> 人工序列(artificial sequence)
<400> 14
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Ser
20 25 30
Ala Met Gly Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
35 40 45
Glu Phe Val Ala Thr Ile Thr Trp Ser Ser Gly Ser Ser Phe Tyr Ala
50 55 60
Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
65 70 75 80
Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Ser Arg Lys Leu Gly Gly Val Val Thr Val Val Thr
100 105 110
Ser Tyr Asp Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 15
<211> 381
<212> DNA
<213> 人工序列(artificial sequence)
<400> 15
caggtgcagc tgcaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg caccttcaat aactcggcca tgggcgccat gggatggttc 120
cgccaggcgc cagggaaaga gcgtgagttt gtcgcgacaa ttacctggag tagtggtagc 180
tcattttatg caaactccgt gaagggccga ttcaccatct ccagagacaa cgccaagaac 240
acggtgtatc tgcaaatgaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca 300
tcacgcaaat tgggaggggt tgtaacggta gttacttcgt atgacttctg gggccagggg 360
acccaggtca ccgtctccag t 381
<210> 16
<211> 13
<212> PRT
<213> 人工序列(artificial sequence)
<400> 16
Gly Arg Thr Phe Asp Asn Tyr Ala Met Gly Ala Met Gly
1 5 10
<210> 17
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 17
Thr Ile Thr Trp Ser Ser Gly Ser Ser Phe Tyr Ala Asn Ser Val Lys
1 5 10 15
Gly
<210> 18
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 18
Ala Ser Arg Lys Leu Gly Gly Val Val Thr Val Val Thr Ser Tyr Asp
1 5 10 15
Phe
<210> 19
<211> 127
<212> PRT
<213> 人工序列(artificial sequence)
<400> 19
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asp Asn Tyr
20 25 30
Ala Met Gly Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
35 40 45
Glu Phe Val Ala Thr Ile Thr Trp Ser Ser Gly Ser Ser Phe Tyr Ala
50 55 60
Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
65 70 75 80
Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Ser Arg Lys Leu Gly Gly Val Val Thr Val Val Thr
100 105 110
Ser Tyr Asp Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 20
<211> 381
<212> DNA
<213> 人工序列(artificial sequence)
<400> 20
caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggacg caccttcgat aactatgcca tgggcgccat gggatggttc 120
cgccaggcgc cagggaaaga gcgtgagttt gtcgcgacaa ttacctggag tagtggtagc 180
tcattttatg caaactccgt gaagggccga ttcaccatct ccagagacaa cgccaagaac 240
acggtgtatc tgcaaatgaa cagcctgaaa cctgacgaca cggccgttta ttactgtgca 300
tcacgcaaat tgggaggggt tgtaacggta gttacttcgt atgacttctg gggccagggg 360
acccaggtca ccgtctccag t 381
<210> 21
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 21
Gly Arg Thr Phe Ser Thr Tyr Ala Val Gly
1 5 10
<210> 22
<211> 18
<212> PRT
<213> 人工序列(artificial sequence)
<400> 22
Gly Arg Leu Thr Trp Ser Gly Ser Arg Thr Tyr Tyr Ala Asp Ser Val
1 5 10 15
Lys Gly
<210> 23
<211> 20
<212> PRT
<213> 人工序列(artificial sequence)
<400> 23
Ala Ala Asp Tyr Arg Ser Asn Ser Thr Trp Ser Leu Gln Ser Pro Ala
1 5 10 15
Arg Tyr Glu Asn
20
<210> 24
<211> 127
<212> PRT
<213> 人工序列(artificial sequence)
<400> 24
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Gly Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Ser Thr Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val
35 40 45
Gly Arg Leu Thr Trp Ser Gly Ser Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Tyr Arg Ser Asn Ser Thr Trp Ser Leu Gln Ser Pro Ala
100 105 110
Arg Tyr Glu Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 25
<211> 381
<212> DNA
<213> 人工序列(artificial sequence)
<400> 25
caggtgcagc tgcaggagtc tgggggagga ttggtgcagg ctggggactc tctgggactc 60
tcctgtacag cctctggacg caccttcagt acctatgccg tggggtggtt ccgccaggct 120
ccagggaagg ggcgtgaatt tgtaggacgt cttacatgga gcgggagtag aacatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240
ctgcaaatga acagcctgag acctgaggac acggccgttt attactgtgc agccgactac 300
cgaagtaaca gtacctggtc cctgcaaagc ccggcacgtt atgaaaattg gggccagggg 360
acccaggtca ccgtctccag t 381
<210> 26
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 26
Gly Arg Thr Val Ser Asn Tyr Ala Met Gly
1 5 10
<210> 27
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 27
Arg Ile Thr Gly Ser Gly Ser Ser Thr Phe Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 28
<211> 19
<212> PRT
<213> 人工序列(artificial sequence)
<400> 28
Ala Ala Asp Arg Trp Arg Ser Met Val Thr Arg Ser Asp Pro Arg Glu
1 5 10 15
Tyr Glu Asn
<210> 29
<211> 126
<212> PRT
<213> 人工序列(artificial sequence)
<400> 29
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Val Ser Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Arg Ile Thr Gly Ser Gly Ser Ser Thr Phe Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Leu Ser Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Arg Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Trp Arg Ser Met Val Thr Arg Ser Asp Pro Arg Glu
100 105 110
Tyr Glu Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 30
<211> 378
<212> DNA
<213> 人工序列(artificial sequence)
<400> 30
caggtgcagc tgcaggagtc tggaggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgtag cctctggacg caccgtcagt aactatgcca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcacgg attaccggga gtggtagtag cacattctat 180
gcagactccg tgaagggccg attcaccatc tccagaaaca acttgtcgaa cacggtgtat 240
ctgcagatga acagcctgaa acgtgaggac acggccgttt attactgtgc agcagatcgc 300
tggcgttcaa tggtgactag atctgacccg agggagtatg agaactgggg ccaggggacc 360
caggtcaccg tctccagt 378
<210> 31
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 31
Gly Arg Thr Val Ser Asn Tyr Ala Met Gly
1 5 10
<210> 32
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 32
Arg Ile Thr Gly Ser Gly Ser Ser Thr Phe Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 33
<211> 19
<212> PRT
<213> 人工序列(artificial sequence)
<400> 33
Ala Ala Asp Arg Trp Arg Ser Met Val Thr Arg Ser Asp Pro Arg Glu
1 5 10 15
Tyr Glu Asn
<210> 34
<211> 126
<212> PRT
<213> 人工序列(artificial sequence)
<400> 34
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Val Ser Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Arg Ile Thr Gly Ser Gly Ser Ser Thr Phe Tyr Ala Asp Ser Val
50 55 60
Lys Gly Leu Phe Thr Ile Ser Arg Asn Asn Leu Ser Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Arg Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Trp Arg Ser Met Val Thr Arg Ser Asp Pro Arg Glu
100 105 110
Tyr Glu Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 35
<211> 378
<212> DNA
<213> 人工序列(artificial sequence)
<400> 35
caggtgcagc tgcaggagtc tggaggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgtag cctctggacg caccgtcagt aactatgcca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcacgg attaccggga gtggtagtag cacattctat 180
gcagactccg tgaagggcct attcaccatc tccagaaaca acttgtcgaa cacggtgtat 240
ctgcagatga acagcctgaa acgtgaggac acggccgttt attactgtgc agcagatcgc 300
tggcgttcaa tggtgactag atctgacccg agggagtatg agaactgggg ccaggggacc 360
caggtcaccg tctccagt 378
<210> 36
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 36
Gly Arg Thr Val Ser Asn Tyr Ala Met Gly
1 5 10
<210> 37
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 37
Arg Ile Thr Gly Ser Gly Ser Ser Thr Phe Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 38
<211> 19
<212> PRT
<213> 人工序列(artificial sequence)
<400> 38
Ala Ala Asp Arg Trp Arg Ser Met Val Thr Arg Ser Tyr Pro Arg Glu
1 5 10 15
Tyr Glu Asn
<210> 39
<211> 126
<212> PRT
<213> 人工序列(artificial sequence)
<400> 39
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Val Ser Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Arg Ile Thr Gly Ser Gly Ser Ser Thr Phe Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Trp Arg Ser Met Val Thr Arg Ser Tyr Pro Arg Glu
100 105 110
Tyr Glu Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 40
<211> 378
<212> DNA
<213> 人工序列(artificial sequence)
<400> 40
caggtgcagc tgcaggagtc tggaggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgtag cctctggacg caccgtcagt aactatgcca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcacgg attaccggga gtggtagtag cacattctat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cgcggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agcagatcgc 300
tggcgttcaa tggtgactag atcttacccg agggagtatg agaactgggg ccaggggacc 360
caggtcaccg tctccagt 378
<210> 41
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 41
Gly Arg Thr Val Ser Asn Tyr Ala Met Gly
1 5 10
<210> 42
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 42
Arg Ile Thr Gly Ser Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 43
<211> 19
<212> PRT
<213> 人工序列(artificial sequence)
<400> 43
Ala Ala Asp Arg Trp Arg Ser Met Val Thr Arg Ser Asp Pro Arg Glu
1 5 10 15
Tyr Glu Asn
<210> 44
<211> 126
<212> PRT
<213> 人工序列(artificial sequence)
<400> 44
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Val Ser Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Arg Ile Thr Gly Ser Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Leu Ser Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Arg Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Trp Arg Ser Met Val Thr Arg Ser Asp Pro Arg Glu
100 105 110
Tyr Glu Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 45
<211> 378
<212> DNA
<213> 人工序列(artificial sequence)
<400> 45
caggtgcagc tgcaggagtc tggaggaggt gtggtgcagg ctggggactc tctgagactc 60
tcctgtgtag cctctggacg caccgtcagt aactatgcca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcacgg attaccggga gtggtcgtac cacatactat 180
gcagactccg tgaagggccg attcaccatc tccagaaaca acttgtcgaa cacggtgtat 240
ctgcagatga acagcctgaa acgtgaggac acggccgttt attactgtgc agcagatcgc 300
tggcgttcaa tggtgactag atctgacccg agggagtatg agaactgggg ccaggggacc 360
caggtcaccg tctccagt 378
<210> 46
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 46
Gly Arg Thr Val Ser Asn Tyr Ala Met Gly
1 5 10
<210> 47
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 47
Arg Ile Thr Gly Ser Gly Ser Ser Thr Phe Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 48
<211> 19
<212> PRT
<213> 人工序列(artificial sequence)
<400> 48
Ala Ala Asp Arg Trp Arg Ser Met Val Thr Arg Ser Asp Pro Arg Asp
1 5 10 15
Tyr Glu Asn
<210> 49
<211> 126
<212> PRT
<213> 人工序列(artificial sequence)
<400> 49
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Val Ser Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Arg Ile Thr Gly Ser Gly Ser Ser Thr Phe Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Leu Ser Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Arg Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Trp Arg Ser Met Val Thr Arg Ser Asp Pro Arg Asp
100 105 110
Tyr Glu Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 50
<211> 378
<212> DNA
<213> 人工序列(artificial sequence)
<400> 50
caggtgcagc tgcaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgtag cctctggacg caccgtcagt aactatgcca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcacgg attaccggga gtggtagtag cacattctat 180
gcagactccg tgaagggccg attcaccatc tccagaaaca acttgtcgaa cacggtgtat 240
ctgcagatga acagcctgaa acgtgaggac acggccgttt attactgtgc agcagatcgc 300
tggcgttcaa tggtgactag atctgacccg agggattatg agaactgggg ccaggggacc 360
caggtcaccg tctccagt 378
<210> 51
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 51
Gly Arg Thr Phe Ser Arg Tyr Ala Val Gly
1 5 10
<210> 52
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 52
Ala Ile Thr Trp Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 53
<211> 15
<212> PRT
<213> 人工序列(artificial sequence)
<400> 53
Ala Val Asp Thr Arg Asn Val Ile Gly Pro Arg Ala Gly Asp Tyr
1 5 10 15
<210> 54
<211> 122
<212> PRT
<213> 人工序列(artificial sequence)
<400> 54
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Leu Gly Arg Asp Phe Val
35 40 45
Ala Ala Ile Thr Trp Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asp Thr Arg Asn Val Ile Gly Pro Arg Ala Gly Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 55
<211> 366
<212> DNA
<213> 人工序列(artificial sequence)
<400> 55
caggtgcagc tgcaggagtc tggaggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg caccttcagt aggtatgccg tgggctggtt ccgccaggct 120
ccagggctgg ggcgtgactt tgtagcagct attacctgga gtggtggtta cacatactat 180
gcggactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240
ttgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agtcgatacg 300
aggaatgtaa tcggcccaag agcgggagac tactggggcc aggggaccca ggtcaccgtc 360
tccagt 366
<210> 56
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 56
Gly Ser Thr Phe Ser Arg Tyr Ala Val Gly
1 5 10
<210> 57
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 57
Ala Ile Thr Trp Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 58
<211> 15
<212> PRT
<213> 人工序列(artificial sequence)
<400> 58
Ala Val Asp Thr Arg Asn Val Ile Gly Pro Arg Ala Gly Asp Tyr
1 5 10 15
<210> 59
<211> 122
<212> PRT
<213> 人工序列(artificial sequence)
<400> 59
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Arg Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Leu Gly Arg Asp Phe Val
35 40 45
Ala Ala Ile Thr Trp Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asp Thr Arg Asn Val Ile Gly Pro Arg Ala Gly Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 60
<211> 366
<212> DNA
<213> 人工序列(artificial sequence)
<400> 60
caggtgcagc tgcaggagtc tggaggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggaag caccttcagt aggtatgccg tgggctggtt ccgccaggct 120
ccagggctgg ggcgtgactt tgtagcagct attacctgga gtggtggtta cacatactat 180
gcggactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240
ttgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agtcgatacg 300
aggaatgtaa tcggcccaag agcgggagac tactggggcc aggggaccca ggtcaccgtc 360
tccagt 366
<210> 61
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 61
Gly Arg Thr Phe Ser Arg Tyr Ala Val Gly
1 5 10
<210> 62
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 62
Ala Ile Thr Trp Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 63
<211> 15
<212> PRT
<213> 人工序列(artificial sequence)
<400> 63
Ala Val Asp Thr Arg Asn Val Ile Gly Pro Arg Ala Gly Asp Tyr
1 5 10 15
<210> 64
<211> 122
<212> PRT
<213> 人工序列(artificial sequence)
<400> 64
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Leu Gly Arg Asp Phe Val
35 40 45
Ala Ala Ile Thr Trp Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Asn Val Glu Asp Thr Gly Val Tyr Tyr Cys
85 90 95
Ala Val Asp Thr Arg Asn Val Ile Gly Pro Arg Ala Gly Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 65
<211> 366
<212> DNA
<213> 人工序列(artificial sequence)
<400> 65
caggtgcagc tgcaggagtc tggaggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg caccttcagt aggtatgccg tgggctggtt ccgccaggct 120
ccagggctgg ggcgtgactt tgtagcagct attacctgga gtggtggtta cacatactat 180
gcggactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgatctat 240
ctccaaatga acagcctgaa cgttgaggac acgggcgttt attactgcgc agtcgatacg 300
aggaatgtaa tcggcccaag agcgggagac tactggggcc aggggaccca ggtcaccgtc 360
tccagt 366
<210> 66
<211> 13
<212> PRT
<213> 人工序列(artificial sequence)
<400> 66
Ala Ala Ser Gly Arg Thr Phe Ser Arg Phe Ala Met Gly
1 5 10
<210> 67
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 67
Ala Ile Ser Trp Ser Gly Gly Met Ile Tyr Tyr Thr Asp Ser Val Lys
1 5 10 15
Gly
<210> 68
<211> 15
<212> PRT
<213> 人工序列(artificial sequence)
<400> 68
Ala Val Asp Thr Arg Asn Val Ile Gly Pro Arg Ala Gly Asp Tyr
1 5 10 15
<210> 69
<211> 122
<212> PRT
<213> 人工序列(artificial sequence)
<400> 69
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Phe
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Ser Gly Gly Met Ile Tyr Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asp Thr Arg Asn Val Ile Gly Pro Arg Ala Gly Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 70
<211> 366
<212> DNA
<213> 人工序列(artificial sequence)
<400> 70
caggtgcagc tgcaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg cactttcagt aggtttgcca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagccgct attagctgga gtggtggtat gatatactat 180
acagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa catgctgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agtcgatacg 300
aggaatgtaa tcggcccaag agcgggagac tactggggcc aggggaccca ggtcaccgtc 360
tccagt 366
<210> 71
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 71
Gly Arg Ala Phe Ser Val Tyr Pro Met Ala
1 5 10
<210> 72
<211> 16
<212> PRT
<213> 人工序列(artificial sequence)
<400> 72
Arg Leu Thr Tyr Thr Ser Asn Thr Phe Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 73
<211> 19
<212> PRT
<213> 人工序列(artificial sequence)
<400> 73
Ala Val Glu Asn Arg Ser Ser Ser Trp Ser Leu Gln Ser Pro Ala Arg
1 5 10 15
Tyr Asp Asp
<210> 74
<211> 125
<212> PRT
<213> 人工序列(artificial sequence)
<400> 74
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Ala Phe Ser Val Tyr
20 25 30
Pro Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ile
35 40 45
Ala Arg Leu Thr Tyr Thr Ser Asn Thr Phe Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Val Glu Asn Arg Ser Ser Ser Trp Ser Leu Gln Ser Pro Ala Arg Tyr
100 105 110
Asp Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 75
<211> 375
<212> DNA
<213> 人工序列(artificial sequence)
<400> 75
caggtgcagc tgcaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcatgtacag cctctggacg cgccttcagt gtctacccca tggcctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tatagcacgt cttacgtata ctagtaacac attctatgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240
cagatgaaca gcctgaaacc tgaggacacg gccgtttatt actgtgcggt cgagaaccgc 300
agtagtagtt ggtccctgca aagcccggca cgttatgatg actggggcca ggggacccag 360
gtcaccgtct ccagt 375
<210> 76
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 76
Gly Arg Thr Gly Ser Arg Tyr Ala Val Gly
1 5 10
<210> 77
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 77
Ala Ile Thr Trp Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 78
<211> 15
<212> PRT
<213> 人工序列(artificial sequence)
<400> 78
Ala Val Asp Thr Arg Asn Val Ile Gly Pro Arg Ala Gly Asp Tyr
1 5 10 15
<210> 79
<211> 122
<212> PRT
<213> 人工序列(artificial sequence)
<400> 79
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Gly Ser Arg Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Leu Gly Arg Asp Phe Val
35 40 45
Ala Ala Ile Thr Trp Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asp Thr Arg Asn Val Ile Gly Pro Arg Ala Gly Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 80
<211> 366
<212> DNA
<213> 人工序列(artificial sequence)
<400> 80
caggtgcagc tgcaggagtc tggaggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg caccggcagt aggtatgccg tgggctggtt ccgccaggct 120
ccagggctgg ggcgtgactt tgtagcagct attacctgga gtggtggtta cacatactat 180
gcggactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgatgtat 240
ctgcaaatga acagcctaaa acctgaagac acggccgttt attactgtgc agtcgatacg 300
aggaatgtaa tcggcccaag agcgggagac tactggggcc aggggaccca ggtcaccgtc 360
tccagt 366
<210> 81
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 81
Ser Ile Asn Ser Gly Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 82
<211> 115
<212> PRT
<213> 人工序列(artificial sequence)
<400> 82
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Asn Ser Gly Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 83
<211> 345
<212> DNA
<213> 人工序列(artificial sequence)
<400> 83
gaggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgcgactc 60
tcctgtgcag cctctggatt caccttcagt agctactgga tgtattggct ccgtcaggct 120
ccagggaagg ggctcgagtg ggtctcatct attaatagtg gtagtagtag cacatactat 180
cgagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctgcaaatga acagtctgaa atctgaggac acggccgtgt attactgtgc aaaagatcct 300
ggtgggtacg ccaaaggcca ggggacccag gtcaccgtct ccagt 345
<210> 84
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 84
Ser Ile Ser Ser Ser Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 85
<211> 115
<212> PRT
<213> 人工序列(artificial sequence)
<400> 85
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Ser Ser Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 86
<211> 345
<212> DNA
<213> 人工序列(artificial sequence)
<400> 86
gaggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgcgactc 60
tcctgtgcag cctctggatt caccttcagt agctactgga tgtattggct ccgtcaggct 120
ccagggaagg ggctcgagtg ggtctcatct attagtagta gtagtagtag cacatactat 180
cgagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctgcaaatga acagtctgaa atctgaggac acggccgtgt attactgtgc aaaagatcct 300
ggtgggtacg ccaaaggcca ggggacccag gtcaccgtct ccagt 345
<210> 87
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 87
Ser Ile Gly Ser Ser Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 88
<211> 115
<212> PRT
<213> 人工序列(artificial sequence)
<400> 88
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Gly Ser Ser Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 89
<211> 345
<212> DNA
<213> 人工序列(artificial sequence)
<400> 89
gaggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgcgactc 60
tcctgtgcag cctctggatt caccttcagt agctactgga tgtattggct ccgtcaggct 120
ccagggaagg ggctcgagtg ggtctcatct attggtagta gtagtagtag cacatactat 180
cgagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctgcaaatga acagtctgaa atctgaggac acggccgtgt attactgtgc aaaagatcct 300
ggtgggtacg ccaaaggcca ggggacccag gtcaccgtct ccagt 345
<210> 90
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 90
Ser Ile Tyr Ser Gly Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 91
<211> 115
<212> PRT
<213> 人工序列(artificial sequence)
<400> 91
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Tyr Ser Gly Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 92
<211> 345
<212> DNA
<213> 人工序列(artificial sequence)
<400> 92
gaggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgcgactc 60
tcctgtgcag cctctggatt caccttcagt agctactgga tgtattggct ccgtcaggct 120
ccagggaagg ggctcgagtg ggtctcatct atttacagtg gtagtagtag cacatactat 180
cgagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctgcaaatga acagtctgaa atctgaggac acggccgtgt attactgtgc aaaagatcct 300
ggtgggtacg ccaaaggcca ggggacccag gtcaccgtct ccagt 345
<210> 93
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 93
Ser Ile Asn Asn Ser Asp Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 94
<211> 115
<212> PRT
<213> 人工序列(artificial sequence)
<400> 94
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Asn Ser Asp Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 95
<211> 345
<212> DNA
<213> 人工序列(artificial sequence)
<400> 95
gaggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgcgactc 60
tcctgtgcag cctctggatt caccttcagt agctactgga tgtattggct ccgtcaggct 120
ccagggaagg ggctcgagtg ggtctcatct attaatagtg acagtagtag cacatactat 180
cgagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctgcaaatga acagtctgaa atctgaggac acggccgtgt attactgtgc aaaagatcct 300
ggtgggtacg ccaaaggcca ggggacccag gtcaccgtct ccagt 345
<210> 96
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 96
Ser Ile Asn Ser Gly Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 97
<211> 115
<212> PRT
<213> 人工序列(artificial sequence)
<400> 97
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Gly Ser Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 98
<211> 345
<212> DNA
<213> 人工序列(artificial sequence)
<400> 98
gaggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgcgactc 60
tcctgtgcag cctctggatt caccttcagt agctactgga tgtattggct ccgtcaggct 120
ccagggaagg ggctcgagtg ggtctcatct attagtggta gtagtagtag cacatactat 180
cgagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctgcaaatga acagtctgaa atctgaggac acggccgtgt attactgtgc aaaagatcct 300
ggtgggtacg ccaaaggcca ggggacccag gtcaccgtct ccagt 345
<210> 99
<211> 226
<212> PRT
<213> 人工序列(artificial sequence)
<400> 99
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly
225
<210> 100
<211> 21
<212> PRT
<213> 人工序列(artificial sequence)
<400> 100
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly
20
<210> 101
<211> 136
<212> PRT
<213> 人工序列(artificial sequence)
<400> 101
Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr
1 5 10 15
Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp
20 25 30
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
35 40 45
Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
50 55 60
Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp
65 70 75 80
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
85 90 95
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
100 105 110
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
115 120 125
Glu Tyr Asn Thr Ser Asn Pro Asp
130 135
<210> 102
<211> 498
<212> PRT
<213> 人工序列(artificial sequence)
<400> 102
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Trp Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Asn Ser Asp Ser Ser Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Pro Gly Gly Tyr Ala Lys Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
115 120 125
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
130 135 140
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
145 150 155 160
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
165 170 175
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
180 185 190
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
195 200 205
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
210 215 220
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
225 230 235 240
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
245 250 255
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
260 265 270
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
275 280 285
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
290 295 300
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
305 310 315 320
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
325 330 335
Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
340 345 350
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln
355 360 365
Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val
370 375 380
Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys
385 390 395 400
Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys
405 410 415
Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu
420 425 430
Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His
435 440 445
Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu
450 455 460
Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp
465 470 475 480
Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
485 490 495
Pro Asp
Claims (10)
1.一种抗PD-L1纳米抗体VHH链的互补决定区CDR区,其特征在于,所述VHH链的互补决定区CDR由以下组成:
氨基酸序列如SEQ ID NO:5n+1所示的CDR1;
氨基酸序列如SEQ ID NO:5n+2所示的CDR2,或氨基酸序列与SEQ ID NO:2所示的序列具有大于85%的序列同一性的CDR2;和
氨基酸序列如SEQ ID NO:5n+3所示的CDR3;
其中,各n独立地为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14或15。
2.一种抗PD-L1纳米抗体的VHH链,其特征在于,所述抗PD-L1纳米抗体的VHH链中包括如权利要求1所述的CDR1、CDR2和CDR3。
3.一种抗PD-L1纳米抗体,其特征在于,所述抗PD-L1纳米抗体是针对PD-L1表位的纳米抗体,并且具有如权利要求2所述的抗PD-L1纳米抗体的VHH链。
4.一种多核苷酸,其特征在于,所述多核苷酸编码选自下组的蛋白质:如权利要求1所述的抗PD-L1纳米抗体VHH链的CDR区、如权利要求2所述的抗PD-L1纳米抗体的VHH链,或如权利要求3所述的抗PD-L1纳米抗体。
5.一种纳米抗体融合蛋白,其特征在于,所述纳米抗体融合蛋白从N端到C端具有如式I所示的结构:
Z1-Z2-L-Z3 (式I)
式中,
Z1为如权利要求2所述的抗PD-L1纳米抗体的VHH链;
Z2为免疫球蛋白的Fc段;
L为接头序列;
Z3为免疫调节分子部分。
6.如权利要求5所述的纳米抗体融合蛋白,其特征在于,所述纳米抗体融合蛋白的氨基酸序列如SEQ ID NO:102所示。
7.一种免疫偶联物,其特征在于,所述免疫偶联物含有:
(a)如权利要求2所述的抗PD-L1纳米抗体的VHH链、如权利要求3所述的抗PD-L1纳米抗体,或如权利要求6所述的纳米抗体融合蛋白;和
(b)选自下组的偶联部分:可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
8.如权利要求3所述的抗PD-L1纳米抗体或如权利要求6所述的纳米抗体融合蛋白的用途,其特征在于,用于制备(a)用于检测PD-L1分子的试剂;(b)用于治疗肿瘤的药物。
9.一种药物组合物,其特征在于,包括:
(i)如权利要求1所述的抗PD-L1纳米抗体VHH链的互补决定区CDR、如权利要求2所述的抗PD-L1纳米抗体的VHH链、或如权利要求3所述的抗PD-L1纳米抗体、如权利要求8所述的纳米抗体融合蛋白,或如权利要求9所述的免疫偶联物;以及
(ii)药学上可接受的载体。
10.如权利要求2所述的VHH链、如权利要求3所述的纳米抗体、如权利要求6所述的纳米抗体融合蛋白,或如权利要求7所述的免疫偶联物的用途,其特征在于,被用于制备药剂、试剂、检测板或试剂盒;
其中,所述试剂、检测板或试剂盒用于:检测样品中PD-L1蛋白;
其中,所述药剂用于治疗或预防表达PD-L1蛋白(即PD-L1阳性)的肿瘤。
Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109096396A (zh) * | 2017-06-20 | 2018-12-28 | 华兰生物工程股份有限公司 | 一种抗pd-l1人源化纳米抗体及其应用 |
US20190177416A1 (en) * | 2016-03-18 | 2019-06-13 | Nanomab Technology Limited | Anti-pd-l1 nanobody, coding sequence and use thereof |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106397592A (zh) * | 2015-07-31 | 2017-02-15 | 苏州康宁杰瑞生物科技有限公司 | 针对程序性死亡配体(pd-l1)的单域抗体及其衍生蛋白 |
KR20200106885A (ko) * | 2017-11-17 | 2020-09-15 | 난징 레전드 바이오테크 씨오., 엘티디. | Pd-l1에 대항하는 단일-도메인 항체 및 이의 변이체들 |
CN110003333B (zh) * | 2019-04-12 | 2022-11-18 | 深圳普瑞金生物药业股份有限公司 | 多肽、pd-l1单域抗体、核苷酸序列及试剂盒 |
-
2019
- 2019-09-12 CN CN201910863109.0A patent/CN112480253B/zh active Active
- 2019-09-12 CN CN202211164309.5A patent/CN115947846A/zh active Pending
-
2020
- 2020-09-07 WO PCT/IB2020/058303 patent/WO2021048725A1/en unknown
- 2020-09-07 AU AU2020344237A patent/AU2020344237A1/en active Pending
- 2020-09-07 CN CN202080064136.2A patent/CN114502588A/zh active Pending
- 2020-09-07 JP JP2022516220A patent/JP2022548066A/ja active Pending
- 2020-09-07 US US17/642,171 patent/US20220315658A1/en active Pending
- 2020-09-07 CA CA3150886A patent/CA3150886A1/en active Pending
- 2020-09-07 EP EP20863990.6A patent/EP4028423A4/en active Pending
- 2020-09-07 KR KR1020227011569A patent/KR20220064986A/ko unknown
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190177416A1 (en) * | 2016-03-18 | 2019-06-13 | Nanomab Technology Limited | Anti-pd-l1 nanobody, coding sequence and use thereof |
CN109096396A (zh) * | 2017-06-20 | 2018-12-28 | 华兰生物工程股份有限公司 | 一种抗pd-l1人源化纳米抗体及其应用 |
Non-Patent Citations (2)
Title |
---|
FEI ZHANG,ET AL.: "Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade", 《CELL DISCOVERY》 * |
冯沛然等: "抗人PD-L1单克隆抗体的制备及其应用", 《中国免疫学杂志》 * |
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EP4028423A1 (en) | 2022-07-20 |
WO2021048725A1 (en) | 2021-03-18 |
CA3150886A1 (en) | 2021-03-18 |
CN115947846A (zh) | 2023-04-11 |
EP4028423A4 (en) | 2023-09-20 |
CN114502588A (zh) | 2022-05-13 |
KR20220064986A (ko) | 2022-05-19 |
JP2022548066A (ja) | 2022-11-16 |
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