CN114763384B - 靶向pd-1的单域抗体及其衍生物和用途 - Google Patents
靶向pd-1的单域抗体及其衍生物和用途 Download PDFInfo
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Abstract
本发明公开了一种靶向人程序性死亡因子PD‑1的单域抗体及其人源化变体。所述VHH链包括SEQ ID NO:2所示的CDR1、SEQ ID NO:3所示的CDR2和SEQ IDNO:4所示的CDR3。本发明的靶向PD‑1的单域抗体能够阻断PD‑1与PD‑L1的相互作用,从而具有广泛的生物应用价值和临床应用价值,并为靶向PD‑1的治疗或诊断药物的开发奠定了新的物质基础。
Description
技术领域
本发明涉及生物技术领域。更具体地说,本发明涉及靶向PD-1的单域抗体及其衍生物和用途。
背景技术
PD-1(程序性死亡受体1,Programmed cell death protein 1,PD-1)及其配体PD-L1是肿瘤免疫的重要靶点。PD-1、PD-L1是一对免疫抑制分子,是免疫***防止自身免疫过激的重要组成部分,其通路的激活具有抑制肿瘤免疫应答、诱导肿瘤特异性T细胞凋亡的作用,与肿瘤发展关系密切。利用单克隆抗体阻断PD-1、PD-L1通路***,目前在临床上显示出良好的疗效和安全性,已有多种抗体类药物获批上市,适应症包括黑色素瘤、非小细胞肺癌、晚期肾细胞癌等多种恶性肿瘤,同时很多正在进行的临床试验尝试开发更多新的适应症。
PD-1除了在激活的T细胞、NKT细胞、B细胞和活化的单核细胞表面表达外,在耗竭的T细胞表面也会高表达。而其配体PD-L1除了在B细胞、T细胞、树突状细胞等免疫细胞表达外,还在外周微血管内皮细胞、心脏和肺脏等器官表达。而PD-L2只在巨噬细胞和树突状细胞表面表达。值得注意的是,PD-L1的表达已经在人类肿瘤中被广泛发现,包括人肺癌、黑色素瘤、卵巢癌和结肠癌等。在体外和体内试验中,阻断PD-L1/PD-1通路可以显著增加T细胞的增殖,细胞因子分泌和T细胞对肿瘤细胞的杀伤作用。在动物体内,阻断PD-L1/PD-1通路显著阻断肿瘤生长。使用PD-1抗体阻断PD-1/PD-L1信号通路,显著增强肿瘤特异T细胞的扩增和肿瘤浸润。以上结果证明了PD-1/PD-L1阻断抗体在肿瘤治疗中的重要应用价值。
目前,FDA已经批准三种PD-1抗体用于多种肿瘤的治疗。这些批准的抗体药物在肿瘤病人身上都取得了很好的治疗效果。但是,总体上临床反应率还比较低,一般在20%左右。提高PD-1/PD-L1阻断抗体的疗效的一种策略是采用双特异性抗体或者多靶点融合蛋白,同时阻断包括PD-1/PD-L1通路在内的其他免疫相关或者不相关通路,希望达到协同的更好的治疗效果。基于PD-1/PD-L1通路的双特异性抗体一般采用双链抗体的形式。这种方法制备的双特异性抗体一般分子量较大,使抗体不易浸润到肿瘤组织,很可能影响临床效果。基于双链的双特异性抗体在生产中常存在错配问题,给下游生产纯化造成很大问题。并且目前的PD-1/PD-L1单抗存在用药量大、总体反应率低等问题。
单域抗体(single domain antibody,sdAb)是一类特殊的,只包含一条抗体重链的抗体。和传统双链抗体类似,它可以选择性地与特定抗原结合。单域抗体最早在骆驼科动物中被发现,之后在护士鲨等软骨鱼纲动物中也被发现。单域抗体单个重链抗体可变区(VHH)是能完整结合抗原的单个功能域,只有12-15kDa。VHH结构简单,在与抗原结合时具有高特异性、高亲和力、免疫原性低、渗透性好以及在进行肿瘤治疗时具有接触到不能被常规抗体接触的较为隐蔽靶点的能力等优点。此外,因为单域抗体只有一条链,所以不会产生双链抗体融合时的错配问题。
发明内容
本发明的目的在于提供一种靶向PD-1的特异性单域抗体。
本发明的目的还在于提供一种靶向PD-1的特异性人源化单域抗体。
本发明的目的还在于提供所述单域抗体或人源化单域抗体在***或制备***的药物中的用途。
在第一方面,本发明提供一种靶向PD-1的单域抗体的VHH链,所述VHH链包括SEQID NO:2所示的CDR1、SEQ ID NO:3所示的CDR2和SEQ ID NO:4所示的CDR3。
在优选的实施方式中,所述PD-1为人PD-1。
在优选的实施方式中,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/或取代至少一个(如1-3个,较佳地1-2个,更佳地1个)氨基酸并能保留与PD-1高亲和力结合、阻断PD-L1与PD-1结合的衍生序列。
在优选的实施方式中,所述VHH链还包括框架区FR1、FR2、FR3和FR4,所述的FR1、FR2、FR3和FR4如SEQ ID NO:1所示的氨基酸序列的1-25位、36-49位、67-98位和110-120位所示。
在优选的实施方式中,所述靶向PD-1的单域抗体的VHH链的氨基酸序列如SEQ IDNO:1所示。
在第二方面,本发明提供一种靶向PD-1的抗体的重链可变区,所述的重链可变区包括SEQ ID NO:2所示的CDR1,SEQ ID NO:3所示的CDR2和SEQ ID NO:4所示的CDR3。
在优选的实施方式中,所述靶向PD-1的抗体的重链可变区的氨基酸序列如SEQ IDNO:1所示。
在第三方面,本发明提供一种靶向PD-1的单域抗体,其具有第一方面所述的VHH链。
在第四方面,本发明提供一种人源化的靶向PD-1的单域抗体的VHH链,以第一方面所述的VHH链为基础对框架区FR1、FR2、FR3和FR4进行人源化。
在优选的实施方式中,所述人源化的靶向PD-1的单域抗体的VHH链的框架区FR1、FR2、FR3和FR4分别如SEQ ID NO:14、或SEQ ID NO:16、或SEQ ID NO:18、或SEQ ID NO:20、或SEQ ID NO:22、或SEQ ID NO:24所示氨基酸序列的1-25位、36-49位、67-98位和110-120位所示。
在优选的实施方式中,所述人源化的靶向PD-1的单域抗体的VHH链氨基酸序列分别如SEQ ID NO:14、或SEQ ID NO:16、或SEQ ID NO:18、或SEQ ID NO:20、或SEQ ID NO:22、或SEQ ID NO:24所示。
在第五方面,本发明提供一种靶向PD-1的抗体,所述抗体包括一个或多个第一方面所述的靶向PD-1的单域抗体的VHH链或第四方面所述的人源化的靶向PD-1的单域抗体的VHH链。
在优选的实施方式中,所述的靶向PD-1的抗体包括单体、二价抗体、和/或多价抗体。
在第六方面,本发明提供一种双特异性抗体,所述双特异性抗体包括第一抗体和第二抗体,所述第一抗体包括第一方面所述的靶向PD-1的单域抗体的VHH链、或第二方面所述的靶向PD-1的抗体的重链可变区、或第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、或第五方面所述的靶向PD-1的抗体。
在优选的实施方式中,所述第二抗体可以结合与第一抗体相同或不同的抗原,或结合与第一抗体相同抗原的不同表位。
在优选的实施方式中,所述第二抗体是单域抗体、单链抗体或双链抗体。
在优选的实施方式中,所述的双特异性抗体包括2-4个靶向PD-1的单域抗体;较佳地,包括2个靶向PD-1的单域抗体;更佳地,所述2个靶向PD-1的单域抗体形成靶向PD-1的单域抗体二聚体。
在第七方面,本发明提供一种融合蛋白,所述融合蛋白包括第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、或第五方面所述的靶向PD-1的抗体,任选的接头序列以及免疫球蛋白的Fc片段。
在优选的实施方式中,所述免疫球蛋白是是IgG1、IgG2、IgG3、IgG4;优选IgG4。
在第八方面,本发明提供一种核酸分子,所述核酸分子编码第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体或第七方面所述的融合蛋白。
在第九方面,本发明提供一种表达载体,所述表达载体包含第八方面所述的核酸分子。
在第十方面,本发明提供一种宿主细胞,所述宿主细胞包含第九方面所述的表达载体,或者其基因组上整合有第八方面所述的核酸分子。
在第十一方面,本发明提供一种制备第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、或第七方面所述的融合蛋白的方法,所述方法包括以下步骤:
1)在适合的条件下,培养如第十方面所述的宿主细胞,从而获得含有所述的靶向PD-1的单域抗体的VHH链、靶向PD-1的抗体的重链可变区、靶向PD-1的单域抗体、人源化的靶向PD-1的单域抗体的VHH链、靶向PD-1的抗体、双特异性抗体或融合蛋白的培养物;以及
2)任选地,从所述培养物中分离或回收所述的靶向PD-1的单域抗体的VHH链、靶向PD-1的抗体的重链可变区、靶向PD-1的单域抗体、人源化的靶向PD-1的单域抗体的VHH链、靶向PD-1的单域抗体、双特异性抗体或融合蛋白。
在第十二方面,本发明提供一种免疫偶联物,所述免疫偶联物含有:
1)如第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体或第七方面所述的融合蛋白;和
2)选自以下的偶联部分:可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在优选的实施方式中,所述偶联部分为药物或毒素。
在优选的实施方式中,所述免疫偶联物是抗体-药物偶联物(Antibody-Drug-Conjugate,ADC)。
在优选的实施方式中,所述偶联部分为可检测标记物。
在优选的实施方式中,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。
在优选的实施方式中,所述免疫偶联物含有:多价(如二价)的如权利要求1所述的靶向PD-1的单域抗体的VHH链、权利要求2所述的靶向PD-1的抗体的重链可变区、权利要求3所述的靶向PD-1的单域抗体、权利要求4所述的人源化的靶向PD-1的单域抗体的VHH链、权利要求5所述的靶向PD-1的抗体、权利要求6所述的双特异性抗体或权利要求7所述的融合蛋白。
在优选的实施方式中,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部分。
在第十三方面,本发明提供一种药物组合物,所述药物组合物包含治疗或诊断有效量的第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、第七方面所述的融合蛋白或第十二方面所述的免疫偶联物,和任选的药学上可接受的赋形剂。
在优选的实施方式中,所述的药物组合物用于***,所述肿瘤选自下组:胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、***癌、结直肠癌、乳腺癌、大肠癌、***癌、***、淋巴癌、肾上腺肿瘤、或***。
在第十四方面,本发明提供第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、第七方面所述的融合蛋白或第十二方面所述的免疫偶联物的用途,用于制备以下试剂:
1)检测PD-1的试剂;
2)阻断PD-1与PD-L1结合的试剂;
3)***的药物。
在优选的实施方式中,所述肿瘤选自下组:胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、***癌、结直肠癌、乳腺癌、大肠癌、***癌、***、淋巴癌、肾上腺肿瘤、或***。
在第十五方面,本发明提供一种试剂盒,所述试剂盒中包括:
1)第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、第七方面所述的融合蛋白、第十二方面所述的免疫偶联物或第十三方面所述的药物组合物;
2)容器;和
3)任选的使用说明书。
在第十六方面,本发明提供一种制备靶向免疫检查点的抗体的方法,所述方法包括以下步骤:
a)利用表达所述免疫检查点的免疫细胞免疫动物;和
b)从步骤1)获得的经免疫动物中获得靶向免疫检查点的抗体。
在优选的实施方式中,所述免疫细胞是原代免疫细胞。
在优选的实施方式中,所述方法还包括先活化免疫细胞的步骤。
在优选的实施方式中,所述免疫检查点包括但不限于:PD-1、CTLA-4、TIM3、LAG3、KIR、GITR、VISTA、4-1BB、CD28、OX40、ICOS等。
在优选的实施方式中,所述免疫细胞是淋巴细胞或吞噬细胞;优选地,所述免疫细胞是T淋巴细胞、B淋巴细胞、K淋巴细胞、NK淋巴细胞、浆细胞、粒细胞、肥大细胞、抗原呈递细胞或单核吞噬细胞***的细胞(例如巨噬细胞);更优选T淋巴细胞。
在优选的实施方式中,所述抗体是多克隆抗体、单克隆抗体、单域抗体;优选单域抗体。
在优选的实施方式中,所述动物包括但不限于小鼠、大鼠、骆驼、羊驼、骆驼、家兔;优选小鼠、羊驼。
在第十七方面,本发明提供一种检测样品中PD-1蛋白的方法,所述方法包括步骤:
1)将待测样品与第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、第七方面所述的融合蛋白或第十二方面所述的免疫偶联物接触;
2)检测是否形成抗原-抗体复合物,如果形成复合物就表示样品中存在PD-1蛋白。
在第十八方面,本发明提供一种治疗疾病的方法,所述方法包括,给有此需要的对象施用治疗有效量的第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、第七方面所述的融合蛋白、第十二方面所述的免疫偶联物或第十三方面所述的药物组合物。
在优选的实施方式中,所述的对象包括哺乳动物;优选人。
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VHH-Fc融合蛋白LL-VHH01-Fc结合PD-1抗原的亲和力检测结果;
图2显示了VHH-Fc融合蛋白LL-VHH01-Fc阻断PD-L1与PD-1蛋白结合的生物活性检测结果;
图3显示了6种人源化抗体均能很好地结合PD-1抗原;
图4显示了6种人源化抗体在细胞水平可以很好地阻断PD-1和PD-L1信号通路;和
图5显示了3种代表性人源化抗体可以在动物体内很好地控制肿瘤生长。
具体实施方式
发明人经过广泛而深入的研究,出乎意料地发现一类靶向PD-1的单域抗体。本发明的单域抗体与PD-1分子有较好的结合活性,同时能够阻断PD-1与PD-L1的相互作用,具有良好的抗肿瘤活性。在该靶向PD-1的单域抗体的基础上,本发明人进一步开发了一系列人源化单域抗体,结果发现这些人源化单域抗体的亲和力、生物学效果与母本抗体类似或者更好,并且具有很好的化学稳定性和血清稳定性。在此基础上完成了本发明。
术语定义
本文所用的术语具有与本领域技术人员常规理解的相同或相似的含义。为清晰起见,对其中的一些术语定义如下。
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。
单域抗体
在本文中,“单域抗体”、“单结构域抗体”、“纳米抗体”等具有相同或相似的含义,均是指缺失抗体轻链、而只有重链可变区的一类抗体分子。单域抗体是最小抗原结合单元,即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,从而构建仅由一个重链可变区组成的单域抗体(VHH)。
在具体的实施方式中,本发明的靶向PD-1的单域抗体VHH链包括SEQ ID NO:2所示的CDR1、SEQ ID NO:3所示的CDR2和SEQ ID NO:4所示的CDR3。在优选的实施方式中,所述靶向PD-1的单域抗体的VHH链的氨基酸序列如SEQ ID NO:1所示。在所述VHH链中,还包括框架区FR1、FR2、FR3和FR4,它们的氨基酸序列如SEQ ID NO:1所示的氨基酸序列的1-25位、36-49位、67-98位和110-120位所示。
在本发明的靶向PD-1的单域抗体VHH链的基础上,本发明人还对该VHH链进行了人源化,从而得到了人源化的靶向PD-1的单域抗体的VHH链。在具体的实施方式中,本发明的人源化VHH链的氨基酸序列分别如SEQ ID NO:14、或SEQ ID NO:16、或SEQ ID NO:18、或SEQID NO:20、或SEQ ID NO:22、或SEQ ID NO:24所示。在所述人源化的VHH链中,还包括框架区FR1、FR2、FR3和FR4,它们的氨基酸序列分别如SEQ ID NO:14、或SEQ ID NO:16、或SEQ IDNO:18、或SEQ ID NO:20、或SEQ ID NO:22、或SEQ ID NO:24所示氨基酸序列的1-25位、36-49位、67-98位和110-120位所示。
在本发明的靶向PD-1的单域抗体VHH链或人源化的VHH链的基础上,本发明还提供了靶向PD-1的抗体,其包括一个或多个所述的靶向PD-1的单域抗体的VHH链或人源化的靶向PD-1的单域抗体的VHH链。本发明还提供了一种双特异性抗体,所述双特异性抗体包括第一抗体和第二抗体,所述第一抗体可以是本发明的靶向PD-1的单域抗体的VHH链、或人源化的VHH链。本领域技术人员可以按照实际需要选择所述双特异性抗体中的第二抗体。例如,所述第二抗体可以结合与第一抗体相同或不同的抗原;如果第二抗体结合与第一抗体相同的抗原,则优选结合在不同的表位上。在具体的实施方式中,所述第二抗体可以是单域抗体,也可以是单链抗体或双链抗体。
本领域技术人员还可以将本发明的靶向PD-1的单域抗体VHH链或人源化的VHH链制成融合蛋白,例如制备成进一步包含免疫球蛋白的Fc片段的融合蛋白。如此得到的融合蛋白不仅具备单域抗体VHH链本身的生物学活性,还能够具备免疫球蛋白的Fc片段所赋予的其它特性,例如血浆半衰期延长、免疫原性降低、稳定性提高等等。在具体的实施方式中,所述融合蛋白包括本发明的靶向PD-1的单域抗体的VHH链或人源化的VHH链,任选的接头序列以及免疫球蛋白的Fc片段。在具体的实施方式中,所述免疫球蛋白是是IgG1、IgG2、IgG3、IgG4;优选IgG4。
本发明不仅包括完整的抗体,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技术人员公知的范围。
本发明抗体指具有PD-1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、***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。
除了几乎全长的多肽外,本发明还包括了本发明单域抗体的片段。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.生物毒素;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.药激活酶(例如,DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL));9.治疗剂(例如,顺铂)或任何形式的纳米颗粒等。
免疫检查点
在本文中,免疫检查点(checkpoint)或免疫检查点分子具有相同的含义,并且与本领域技术人员常规理解的相同。其是指在免疫细胞上表达、能调节免疫激活程度的一系列分子,它们对防止自身免疫(免疫功能发生异常,对正常细胞发动攻击)作用的发生起重要作用。因此,“免疫检查点分子”的重要功能之一就是使免疫***的活化保持在正常的范围之内,以防止免疫***的过度活化。
免疫检查点分子的表达和功能异常是很多疾病发生的重要原因之一,比如免疫检查点分子过度表达或功能过强,免疫功能受到抑制,机体的免疫力就低下,人就容易得肿瘤等疾病;反之,免疫检查点分子的这种免疫抑制功能如果太差,机体的免疫功能也会异常。肿瘤细胞会表达一些物质,来激活免疫检查点,后者一旦被激活,则抗原不能被提呈至T细胞,阻断了肿瘤免疫环中的提呈抗原过程,从而抑制T细胞的免疫功能,使得肿瘤细胞逃脱免疫监视,进而存活。
常用的免疫检查点有CTLA-4(细胞毒T淋巴细胞相关抗原-4,cytotoxic Tlymphocyte-associated antigen-4)、PD-1、TIM3、LAG3、KIR、GITR、VISTA、4-1BB等。
免疫细胞
在本文中,免疫细胞和免疫效应细胞具有相同的含义,并且与本领域技术人员常规理解的相同。其是指参与免疫应答或与免疫应答相关的细胞,包括淋巴细胞和吞噬细胞。在具体的实施方式中,所述免疫细胞是指能识别抗原、从而产生特异性免疫应答的淋巴细胞。所述淋巴细胞主要是T淋巴细胞、B淋巴细胞、K淋巴细胞和NK淋巴细胞。除淋巴细胞外,参与免疫应答的细胞还有浆细胞、粒细胞、肥大细胞、抗原呈递细胞及单核吞噬细胞***的细胞(例如巨噬细胞)。
免疫偶联物
本发明还提供一种免疫偶联物,其含有本发明的靶向PD-1的单域抗体的VHH链、人源化的VHH链等以及偶联部分。在具体的实施方式中,所述偶联部分可以是可检测标记物、药物、毒素、细胞因子、放射性核素或酶等,从而实现诊断、检测或治疗等等目的。
在优选的实施方式中,所述免疫偶联物是抗体-药物偶联物(Antibody-Drug-Conjugate,ADC)。
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-1蛋白,从而阻断PD-1和PD-L1的相互作用。因此,本发明的药物组合物可用于***。在优选的实施方式中,所述肿瘤选自下组:胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、***癌、结直肠癌、乳腺癌、大肠癌、***癌、***、淋巴癌、肾上腺肿瘤、或***。此外,本发明的药物组合物还可与其他治疗剂联用。
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%)的本发明上述的单域抗体(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。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。
检测方法
本发明还涉及检测PD-1蛋白的方法。该方法步骤大致如下:将待测样品与本发明的靶向PD-1的单域抗体的VHH链、或人源化的VHH链、抗体、融合蛋白或免疫偶联物等接触;随后检测是否形成抗原-抗体复合物,如果形成复合物就表示样品中存在PD-1蛋白。
试剂盒
本发明还提供了一种含有本发明的靶向PD-1的单域抗体的VHH链、或人源化的VHH链、抗体、融合蛋白或免疫偶联物等的试剂盒。在具体的实施方式中,所述的试剂盒还包括容器、使用说明书、缓冲剂等。
制备靶向免疫检查点的抗体的方法
在研究过程中,本发明人发现直接利用PD-1蛋白免疫动物得到的PD-1单域抗体的活性不佳。然而,当本发明人利用T细胞进行免疫时,获得了活性很好的PD-1单域抗体。
基于上述发现,本发明还提供了一种特殊的免疫方法,该方法能够制备靶向免疫检查点的抗体。所述方法包括利用表达免疫检查点的免疫细胞免疫动物。在优选的实施方式中,可以在利用表达免疫检查点的免疫细胞免疫动物之前先活化所述免疫细胞。活化免疫细胞的方法或技术手段是本领域技术人员熟知的。
本发明的制备靶向免疫检查点的抗体的方法中,利用的免疫细胞优选原代免疫细胞。在本发明的方法中,所免疫的动物包括但不限于小鼠、骆驼、羊驼、大鼠、家兔;优选小鼠、羊驼。本发明的方法获得的抗体可以是多克隆抗体、单克隆抗体、单域抗体。
本发明的优点:
1.本发明的靶向PD-1的单域抗体与PD-1具有良好的结合亲和力,从而能够有效阻断PD-1和PD-L1结合;
2.本发明的人源化单域抗体能够保留与PD-1的良好结合亲和力,并且在体外细胞水平表现出与母本抗体类似、甚至更好的生物学效果;
3.本发明的人源化单域抗体具有很好的化学稳定性和血清稳定性;和
4.本发明的靶向PD-1的单域抗体和人源化单域抗体具有广泛的生物应用价值和临床应用价值,从而为靶向PD-1的治疗或诊断药物的开发奠定了新的物质基础。
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:噬菌体展示免疫库的构建
1.1动物免疫
将冻存的人外周血单个核细胞(购自妙通生物)复苏后,将浓度调整到2*106/ml。然后按照说明书推荐比例加入负载CD3抗体和CD28抗体的微型磁珠(Thermo FisherScientific)以激活PBMCs中的T细胞。激活T细胞以后,使用磁力架去磁珠;之后按照1600rpm,5分钟条件离心收取细胞;使用冻存培养基冻存细胞。冻存培养基为:RPMI-1640:FBS:DMSO=4:5:1。暂时将冻存的细胞储存于液氮中。
选取一只成年健康的羊驼(Alpaca)。首先将冻存的T细胞复苏,然后使用1*PBS洗涤细胞一次。使用缓冲液重悬细胞后对羊驼进行皮下免疫。之后采用相同的方法对羊驼进行五次免疫。
1.2噬菌体文库构建
免疫结束后,采集羊驼四免和五免之后的外周血各50mL,将两者混合后分离外周血单个核细胞(PBMCs)。进一步使用RNAiso Plus试剂(Takara,货号:9109)提取总RNA。使用PrimeScriptTMII 1st Strand cDNA Synthesis Kit(Takara,货号:6210A)试剂盒,按照试剂盒提供的说明书,对提取的总RNA进行反转录。使用巢式PCR扩增羊驼重链可变区的DNA片段。共进行两轮巢式PCR。
将载体(pComb3XSS)与PCR扩增的目的片段分别使用SfiI进行酶切,50℃过夜酶切,然后回收目的片段。将酶切后的PCR产物和载体进行连接,连接摩尔比例为载体:PCR产物=1:3。使用电转化的方法将连接有VHH的载体导入到感受态细胞中,构建重链单域抗体噬菌体展示文库。电转后进行铺板。为检测文库的***率,随机选取板上48个克隆做菌落PCR。结果显示***率达到100%。通过梯度稀释铺板,计算库容的大小为1.0×109。将细菌文库接入2×300mL 2YT+A+G(Amp:100ug/ml、Glu:1%)培养基中,至其初始OD600=0.1-0.2,37℃、230rpm培养至OD600=0.8以上。根据OD600值加入辅助噬菌体M13KO7,(辅助噬菌体:细菌=20:1),制备噬菌体文库。通过梯度稀释噬菌体文库然后铺板,计算克隆数的方法测到噬菌体文库滴度为5.76×1013cfu/mL。
实施例2:抗人PD-1单域抗体筛选
通过固相筛选的方法,对所构建的羊驼免疫库进行亲和力筛选,获得特异性噬菌体文库。
2.1亲和淘选
2.1.1淘选
1)使用pH值为9.6的碳酸盐缓冲液稀释靶抗原,终浓度为5μg/mL。然后按100μL/孔加入到酶标孔中,4℃包被过夜;
2)弃包被液,使用PBS洗涤3次。然后每孔中加入300μL 3%BSA-PBS封闭液,37℃条件封闭1小时;
3)之后弃掉封闭液,使用PBS洗涤3次。加入100μL噬菌体文库,37℃孵育1小时;
4)吸出未结合的噬菌体,用PBST洗涤平板6次,PBS洗涤平板2次;
5)加入100μL的Gly-HCl洗脱液到每个孔中,37℃孵育8min;
6)将洗脱液转移至1.5mL无菌离心管中,迅速用15μL Tris-HCl中和缓冲液中和;
7)取10μL中和后的溶液进行梯度稀释,测定噬菌体滴度,计算淘选回收率。将其余洗脱物混合后进行扩增和纯化,用于下一轮亲和淘选。改变淘选条件,每一轮淘选条件如表1。
表1.亲和淘选条件
2.1.2文库扩增
淘选后需要将文库进行扩增,主要步骤如下:
1)将淘选洗脱物与处于对数生长前期的E.coli TG1培养物5mL混匀,37℃条件静置30min,然后220r/min振荡培养30min;
2)1000g离心15min,去上清,用500μL 2×YT重悬涂布于200mm 2×YT-GA平板;
3)用10ml 2×YT液体培养基刮菌,取500μl悬液加入50ml 2×YT液体培养基中,37℃振摇30min;按cell:phage=1:20的比例加入M13K07辅助噬菌体,37℃静置30min,220r/min振摇培养30min;
4)将培养物分装于离心管中,25℃,5000r/min,10min,细胞沉淀以50mL2×YT-AK液体培养基重悬,30℃,230r/min振荡培养过夜;
5)将过夜培养物4℃,10000r/min离心20min,将上清转移至新离心管,加入1/5体积的PEG-NaCl,混匀后置于4℃2h以上;
6)离心,4℃,10000r/min,20min,去除上清,将沉淀重悬于1mL PBS中,加入1/5体积的PEG/NaCl,混匀后置于4℃1h以上;
7)4℃,12000r/min,2min,去除上清,将沉淀悬浮于200μL PBS中,即为扩增产物,测定滴度,用于下一轮淘选或者分析。
2.1.3噬菌体救援
1)从第二轮淘选洗脱物滴度的平板上用灭菌牙签随机挑取96个单克隆,接种于1mL 2×YT-A中,37℃,220r/min振荡培养8h;
2)取200μL上述培养物,按cell:phage=1:20的比例加入M13K07噬菌体,37℃,静置15min;
3)220r/min振荡培养45min;
4)补加800μL体积的2×YT-AK,30℃,剧烈振荡培养过夜;
5)第二天12000rpm离心2min;
6)取上清,用于单克隆ELISA鉴定。
2.1.4阳性噬菌体克隆鉴定
1)将PD-1抗原用pH值为9.6的碳酸盐缓冲液稀释至终浓度为2μg/mL,按100μL/孔加入酶标孔中,4℃包被过夜;
2)弃包被液,PBST洗涤3次;
3)每孔加入300μL 5%脱脂牛奶,37℃封闭1h;
4)PBST洗涤3次,每孔加入50μL噬菌体培养菌液上清和50μL 5%脱脂牛奶,37℃,孵育1h;
5)PBST洗涤5次,之后加入辣根过氧化物酶标记的抗M13抗体(用5%脱脂牛奶按1:10000稀释),100μL/孔,37℃作用1h;
6)PBST洗板6次。加入TMB显色液显色,100μL/孔,37℃,7min;
7)加入终止液终止反应,50μL/孔,于450nm下测光密度。
2.2阳性克隆的序列确定
结果检测到三个阳性克隆,将三个阳性细菌克隆送苏州泓迅生物技术有限公司进行测序。其中一个克隆序列测序成功。测序结果分析得到抗PD-1抗体VHH区,将该VHH命名为LL-VHH01。使用Kabat命名法对LL-VHH01进行CDR区分析,得到对应的CDR区域序列。LL-VHH01的全长和CDR区的氨基酸序列见表2,核酸序列见表3。
表2.VHH的氨基酸序列和对应的CDR氨基酸序列
LL-VHH01的氨基酸序列为SEQ ID NO:1,CDR1序列为SEQ ID NO:2,CDR2序列为SEQID NO:3、CDR3的序列为SEQ ID NO:4。
表3.LL-VHH01的核酸序列和对应的CDR核酸序列
实施例3:制备PD-1单域抗体VHH-Fc融合蛋白
通过数据库Uniprot获得人免疫球蛋白gamma4(IgG4)的恒定区氨基酸序列(P01861)。
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:9)
将PD-1单域抗体VHH氨基酸序列和hIgG4-Fc氨基酸序列连接,得到PD-1VHH-Fc融合蛋白,命名为:LL-VHH01-Fc。通过基因合成方式得到抗人PD-1单域抗体VHH-Fc融合蛋白LL-VHH01-Fc的基因序列。
然后将PD-1VHH-Fc融合蛋白基因克隆至表达载体pCDNA4(Invitrogen,CatV86220)中。使用瞬时转染HEK293悬浮细胞的方法对PD-1单域抗体VHH-Fc融合蛋白LL-VHH01-Fc进行表达。表达完成后,收取上清液,使用Protein A亲和层析柱纯化,最终获得纯化的PD-1单域抗体的Fc融合蛋白LL-VHH01-Fc。
LL-VHH01-Fc
基因序列为:SEQ ID NO:10
CAGTTGCAGCTCGTGGAGTCGGGAGGAGGGCTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTCCAGTATGTATGCCATGGGCTGGTTCCGCCAGTCTCCAGGGAACGAGCGCGAGTTTGTAGCGGGGATTGGCTGGGAGAATAATACCCCATACTATGCACGCTCCGTGGAGGGCCGATTCACCATCTCCAGAGACAACGTCAAGAACACGGTCTTTCTACAAATGAACAGACTGAAACCTGAGGACGCGGCCGTTTATTTTTGTGCAGCCCAAATCGGAATATCCGGTACATTGGGGGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA。
氨基酸序列为:SEQ ID NO:11
QLQLVESGGGLVQAGGSLRLSCAASGRTSSMYAMGWFRQSPGNEREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVFLQMNRLKPEDAAVYFCAAQIGISGTLGDYWGQGTQVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。
实施例4:制备PD-1阳性对照抗体
通过专利WO2008156712A1,获得Merck Sharp&Dohme(MSD)公司的抗PD-1抗体Pembrolizumab氨基酸序列。其中重链氨基酸序列为SEQ ID NO:12
QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。
轻链氨基酸序列为SEQ ID NO:13
EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
同时参考专利中抗体的表达纯化的方法对Pembrolizumab抗体类似物进行表达纯化。得到MSD公司的抗PD-1抗体类似物,重新命名为LL-Pos。
实施例5:ELISA法检测PD-1单域抗体VHH-Fc融合蛋白对PD-1蛋白的结合能力
首先将人PD-1蛋白(购自北京百普赛斯,货号PD1-H5221)使用PBS(购自Hyclone公司)稀释至1.5μg/mL,按照100μl/孔加入96孔酶标板进行抗原包被,置于37℃恒温培养箱中孵育60min。孵育结束后洗板三次并按照200μl/孔加入含有2%BSA的PBS进行封闭,置于37℃恒温培养箱内孵育60min。同时,将样品LL-VHH01-Fc及对照抗体LL-Pos用稀释液(含有2%BSA的PBS)稀释至100μg/ml,并在样品稀释板上以3倍梯度依次稀释至0.56ng/ml。然后,按照100μl/孔将样品加入到96孔酶标板中并置于37℃恒温培养箱内孵育60min。孵育结束后洗板并将二抗山羊抗-人IgG(Fc特异性)-HRP抗体(购自Sigma公司,货号A-0170)用2%BSA稀释6000倍,按照100μl/孔加入酶标板并置于37℃恒温培养箱内孵育30min。孵育结束后洗板三次并按照100μl/孔加入显色液,显色液为100μg/ml的TMB(3,3',5,5'-四甲基联苯胺),置于37℃恒温培养箱内避光孵育15min。最后,按照100μl/孔加入终止液(2M/L盐酸溶液)并使用酶标仪(Thermo Fisher Scientific,Varioskan LUX)检测450nm/620nm测吸收值,使用Graphpad Prism分析数据。
结果如图1所示,LL-Pos结合PD-1蛋白的EC50为11.84ng/ml。而LL-VHH01-Fc的EC50为29.69ng/ml。在该实验条件下,LL-VHH01-Fc结合PD-1蛋白的量效反应曲线的上平台值明显高于LL-Pos。综合来看,本发明VHH的Fc融合蛋白对PD-1抗原的亲和力与对照抗体LL-Pos基本相当。
实施例6:使用荧光素酶报告基因法检测VHH-Fc融合蛋白的生物学活性
培养CHO/PD-L1细胞(购自Promega公司)后消化并用含有10%FBS的F-12营养混合物完全培养基重悬细胞,根据细胞计数结果使用完全培养基调整细胞密度至5×105个细胞/ml。随后将细胞悬液转移至加样槽中并使用多道移液器以100μl/孔加入到96孔板中,放置于37℃的5%CO2培养箱中培养16~22h。同时,培养Jurkat/PD-1细胞(购自Promega公司)细胞并根据细胞计数结果使用分析培养基(RPMI 1640Medium+2%FBS)调整细胞密度至2×106个细胞/ml。将加入CHO/PD-L1细胞的细胞培养板从培养箱中取出并使用多道移液器每孔取出100μl培养液,随后按照40μl/孔加入梯度稀释(起始浓度为1650nM)的待测样品。然后,将上述Jurkat/PD-1细胞悬液转移至加样槽中并按照40μl/孔加入到细胞培养板中,置于37℃的5%CO2培养箱培养4~6h。培养结束后取出细胞培养板,置于室温放置5~10min,然后每孔加入40μl One-Glo试剂(购自Promega公司,货号E6130),置于混匀器上混匀5~10min,然后使用多功能酶标仪读取化学发光信号值,使用Graphpad Prism分析数据。
结果如图2所示,LL-VHH01-Fc可有效阻断PD-1和PD-L1结合,并激活下游通路产生信号,其量效曲线的EC50为0.35μg/ml。阳性对照LL-Pos也可有效阻断PD-1和PD-L1结合,并激活下游通路产生信号,其量效曲线的EC50为1.09μg/ml。结果表明LL-VHH01-Fc阻断PD-1和PD-L1结合的能力类似于阳性对照。
图1和图2的结果表明本发明的PD-1单域抗体融合蛋白在对抗原PD-1的亲和力和对PD-1和PD-L1结合的阻断作用方面都等同于或者优于已经上市的对照抗体,因此可能在临床上会有更好的效果。另外,单域抗体没有轻链,非常适合作为重要的组成抗体进行双抗或者多特异性抗体的开发,有望开发出疗效更好的抗体药物。
实施例7:单域抗体的人源化
LL-VHH01为羊驼来源抗体,为了提高该单域抗体的成药性,对LL-VHH01进行人源化。基本步骤:
1.使用IMGT数据库进行抗体序列比对。根据数据库序列比对结果,选取IGHV3-23*04作为LL-VHH01人源化母本载体;
2.将LL-VHH01单域抗体的CDR区移植到IGHV3-23*04;
3.对移植后的人源化抗体进行回复突变以确保人源化抗体的亲和力。
得到六个候选人源化抗体:
>huVHH3-1
EVQLVESGGGLVQPGGSLRLSCAASGRTSSMYAMGWFRQAPGKGREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVYLQMNSLRAEDTAVYYCAAQIGISGTLGDYWGQGTQVTVSS(SEQ ID NO:14)
核酸序列:
GAGGTGCAGCTTGTTGAAAGTGGTGGAGGTCTTGTTCAACCAGGGGGCTCCCTCAGACTGTCTTGTGCGGCGAGCGGGCGGACATCCTCTATGTATGCGATGGGTTGGTTCCGACAGGCCCCCGGTAAAGGACGGGAGTTCGTAGCTGGCATCGGTTGGGAAAACAATACCCCTTATTACGCCCGGTCTGTTGAAGGTCGATTTACTATAAGTCGGGACAATGTGAAAAATACTGTCTATCTCCAAATGAACTCTCTGCGGGCCGAAGATACAGCGGTGTACTATTGTGCCGCCCAAATTGGAATCAGCGGAACATTGGGTGATTATTGGGGCCAAGGTACGCAAGTTACAGTCTCCTCA(SEQ ID NO:15)
>huVHH3-2
EVQLVESGGGLVQPGGSLRLSCAASGRTSSMYAMGWFRQAPGNEREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVYLQMNSLRAEDTAVYYCAAQIGISGTLGDYWGQGTQVTVSS(SEQ ID NO:16)
核酸序列:
GAAGTCCAACTGGTCGAAAGCGGCGGCGGTCTCGTCCAACCTGGAGGCTCTCTTAGGTTGTCATGTGCCGCCTCAGGCAGAACATCCAGCATGTACGCAATGGGTTGGTTCAGACAGGCTCCGGGGAACGAGCGAGAATTCGTCGCGGGAATAGGATGGGAGAACAACACCCCATACTACGCACGCAGTGTGGAAGGCCGATTCACTATTAGTCGGGATAATGTTAAAAACACGGTCTACCTTCAAATGAACTCCCTTCGCGCAGAGGATACTGCAGTTTATTATTGCGCGGCCCAAATAGGTATAAGTGGAACACTCGGGGACTACTGGGGCCAGGGAACACAGGTAACCGTATCATCA(SEQ ID NO:17)
>huVHH3-3
EVQLVESGGGLVQPGGSLRLSCAASGRTSSMYAMGWFRQAPGNEREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVFLQMNRLRAEDTAVYYCAAQIGISGTLGDYWGQGTQVTVSS(SEQ ID NO:18)
核酸序列:
GAGGTCCAGTTGGTAGAAAGTGGTGGTGGGTTGGTGCAACCCGGTGGCTCATTGAGGCTGTCTTGTGCTGCGAGTGGCAGGACATCCTCTATGTATGCGATGGGATGGTTCCGACAAGCTCCAGGAAACGAGCGCGAGTTCGTAGCCGGAATTGGTTGGGAAAACAATACGCCCTATTATGCACGGTCTGTCGAGGGGAGGTTCACTATCTCACGCGACAACGTCAAGAACACAGTGTTTCTTCAGATGAACCGACTCCGGGCGGAGGATACGGCCGTATATTATTGCGCAGCGCAAATCGGTATATCCGGCACTCTTGGTGACTATTGGGGCCAGGGTACACAAGTGACAGTCTCTTCA(SEQ ID NO:19)
>huVHH3-4
EVQLVESGGGLVQPGGSLRLSCAASGRTSSMYAMGWFRQAPGNEREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVFLQMNRLKPEDTAVYYCAAQIGISGTLGDYWGQGTQVTVSS(SEQ ID NO:20)
核酸序列:
GAAGTGCAACTCGTGGAGAGCGGGGGCGGACTTGTCCAACCGGGAGGGAGTTTGAGACTCTCATGCGCCGCCTCTGGTAGAACTAGCAGCATGTACGCTATGGGATGGTTCAGGCAGGCTCCAGGGAACGAACGAGAATTCGTTGCAGGCATAGGATGGGAAAACAACACCCCATATTACGCTCGGTCCGTGGAAGGACGATTTACTATAAGCCGGGACAATGTAAAAAATACTGTCTTTCTCCAGATGAATAGGCTCAAGCCGGAGGATACAGCAGTTTATTATTGCGCTGCTCAAATTGGGATTAGCGGGACCCTGGGTGACTATTGGGGGCAGGGAACGCAAGTGACTGTCAGTTCT(SEQ ID NO:21)
>huVHH3-5
ELQLVESGGGLVQAGGSLRLSCAASGRTSSMYAMGWFRQAPGNEREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVFLQMNRLKPEDTAVYYCAAQIGISGTLGDYWGQGTQVTVSS(SEQ ID NO:22)
核酸序列:
GAGTTGCAACTGGTGGAAAGTGGTGGCGGGTTGGTTCAGGCAGGCGGTTCCCTTCGCCTCTCCTGTGCGGCGAGTGGACGCACATCATCCATGTACGCAATGGGGTGGTTTCGACAAGCCCCCGGAAACGAACGCGAATTTGTTGCTGGGATTGGATGGGAAAACAATACGCCGTACTATGCCCGGAGCGTAGAAGGACGATTCACCATTTCCAGGGACAACGTCAAAAACACGGTCTTCTTGCAAATGAACCGCTTGAAACCAGAGGATACCGCAGTATACTATTGTGCTGCCCAGATCGGCATATCAGGCACACTGGGCGACTATTGGGGCCAAGGGACCCAGGTCACTGTATCCAGC(SEQ ID NO:23)
>huVHH3-6
ELQLVESGGGLVQAGGSLRLSCAASGRTSSMYAMGWFRQAPGNEREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVFLQMNRLKPEDAAVYFCAAQIGISGTLGDYWGQGTQVTVSS(SEQ ID NO:24)
核酸序列:
GAACTGCAACTCGTAGAATCTGGGGGTGGCTTGGTCCAGGCCGGGGGCAGTCTGCGACTTTCCTGTGCCGCATCAGGAAGGACCTCCAGCATGTATGCGATGGGATGGTTCCGACAAGCTCCGGGAAATGAGCGCGAGTTTGTTGCGGGAATTGGCTGGGAGAATAACACGCCCTATTATGCTCGGTCCGTAGAGGGGAGGTTCACTATCAGCCGAGATAATGTAAAAAACACCGTATTCCTCCAAATGAATCGGTTGAAACCAGAGGACGCAGCGGTCTACTTTTGCGCCGCGCAAATCGGCATAAGCGGTACATTGGGGGATTACTGGGGTCAAGGCACACAGGTAACCGTCTCTAGT(SEQ ID NO:25)
实施例8.制备人IgG4 Fc标签的六种人源化抗体
参考实施例3的实验流程,表达、纯化融合人IgG4 Fc标签的六种人源化抗体,分别命名为:huVHH3-1-Fc、huVHH3-2-Fc、huVHH3-3-Fc、huVHH3-4-Fc、huVHH3-5-Fc、huVHH3-6-Fc。六种人源化抗PD-1单域抗体融合hIgG4 Fc的序列如下:
>huVHH3-1-Fc
EVQLVESGGGLVQPGGSLRLSCAASGRTSSMYAMGWFRQAPGKGREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVYLQMNSLRAEDTAVYYCAAQIGISGTLGDYWGQGTQVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:26)
核酸序列:
GAGGTGCAGCTTGTTGAAAGTGGTGGAGGTCTTGTTCAACCAGGGGGCTCCCTCAGACTGTCTTGTGCGGCGAGCGGGCGGACATCCTCTATGTATGCGATGGGTTGGTTCCGACAGGCCCCCGGTAAAGGACGGGAGTTCGTAGCTGGCATCGGTTGGGAAAACAATACCCCTTATTACGCCCGGTCTGTTGAAGGTCGATTTACTATAAGTCGGGACAATGTGAAAAATACTGTCTATCTCCAAATGAACTCTCTGCGGGCCGAAGATACAGCGGTGTACTATTGTGCCGCCCAAATTGGAATCAGCGGAACATTGGGTGATTATTGGGGCCAAGGTACGCAAGTTACAGTCTCCTCAGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA(SEQ IDNO:27)
>huVHH3-2-Fc
EVQLVESGGGLVQPGGSLRLSCAASGRTSSMYAMGWFRQAPGNEREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVYLQMNSLRAEDTAVYYCAAQIGISGTLGDYWGQGTQVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:28)
核酸序列:
GAAGTCCAACTGGTCGAAAGCGGCGGCGGTCTCGTCCAACCTGGAGGCTCTCTTAGGTTGTCATGTGCCGCCTCAGGCAGAACATCCAGCATGTACGCAATGGGTTGGTTCAGACAGGCTCCGGGGAACGAGCGAGAATTCGTCGCGGGAATAGGATGGGAGAACAACACCCCATACTACGCACGCAGTGTGGAAGGCCGATTCACTATTAGTCGGGATAATGTTAAAAACACGGTCTACCTTCAAATGAACTCCCTTCGCGCAGAGGATACTGCAGTTTATTATTGCGCGGCCCAAATAGGTATAAGTGGAACACTCGGGGACTACTGGGGCCAGGGAACACAGGTAACCGTATCATCAGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA(SEQ IDNO:29)
>huVHH3-3-Fc
EVQLVESGGGLVQPGGSLRLSCAASGRTSSMYAMGWFRQAPGNEREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVFLQMNRLRAEDTAVYYCAAQIGISGTLGDYWGQGTQVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:30)
核酸序列:
GAGGTCCAGTTGGTAGAAAGTGGTGGTGGGTTGGTGCAACCCGGTGGCTCATTGAGGCTGTCTTGTGCTGCGAGTGGCAGGACATCCTCTATGTATGCGATGGGATGGTTCCGACAAGCTCCAGGAAACGAGCGCGAGTTCGTAGCCGGAATTGGTTGGGAAAACAATACGCCCTATTATGCACGGTCTGTCGAGGGGAGGTTCACTATCTCACGCGACAACGTCAAGAACACAGTGTTTCTTCAGATGAACCGACTCCGGGCGGAGGATACGGCCGTATATTATTGCGCAGCGCAAATCGGTATATCCGGCACTCTTGGTGACTATTGGGGCCAGGGTACACAAGTGACAGTCTCTTCAGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA(SEQ IDNO:31)
>huVHH3-4-Fc
EVQLVESGGGLVQPGGSLRLSCAASGRTSSMYAMGWFRQAPGNEREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVFLQMNRLKPEDTAVYYCAAQIGISGTLGDYWGQGTQVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:32)
核酸序列:
GAAGTGCAACTCGTGGAGAGCGGGGGCGGACTTGTCCAACCGGGAGGGAGTTTGAGACTCTCATGCGCCGCCTCTGGTAGAACTAGCAGCATGTACGCTATGGGATGGTTCAGGCAGGCTCCAGGGAACGAACGAGAATTCGTTGCAGGCATAGGATGGGAAAACAACACCCCATATTACGCTCGGTCCGTGGAAGGACGATTTACTATAAGCCGGGACAATGTAAAAAATACTGTCTTTCTCCAGATGAATAGGCTCAAGCCGGAGGATACAGCAGTTTATTATTGCGCTGCTCAAATTGGGATTAGCGGGACCCTGGGTGACTATTGGGGGCAGGGAACGCAAGTGACTGTCAGTTCTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA(SEQ IDNO:33)
>huVHH3-5-Fc
ELQLVESGGGLVQAGGSLRLSCAASGRTSSMYAMGWFRQAPGNEREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVFLQMNRLKPEDTAVYYCAAQIGISGTLGDYWGQGTQVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:34)
核酸序列:
GAGTTGCAACTGGTGGAAAGTGGTGGCGGGTTGGTTCAGGCAGGCGGTTCCCTTCGCCTCTCCTGTGCGGCGAGTGGACGCACATCATCCATGTACGCAATGGGGTGGTTTCGACAAGCCCCCGGAAACGAACGCGAATTTGTTGCTGGGATTGGATGGGAAAACAATACGCCGTACTATGCCCGGAGCGTAGAAGGACGATTCACCATTTCCAGGGACAACGTCAAAAACACGGTCTTCTTGCAAATGAACCGCTTGAAACCAGAGGATACCGCAGTATACTATTGTGCTGCCCAGATCGGCATATCAGGCACACTGGGCGACTATTGGGGCCAAGGGACCCAGGTCACTGTATCCAGCGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA(SEQ IDNO:35)
>huVHH3-6-Fc
ELQLVESGGGLVQAGGSLRLSCAASGRTSSMYAMGWFRQAPGNEREFVAGIGWENNTPYYARSVEGRFTISRDNVKNTVFLQMNRLKPEDAAVYFCAAQIGISGTLGDYWGQGTQVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:36)
核酸序列:
GAACTGCAACTCGTAGAATCTGGGGGTGGCTTGGTCCAGGCCGGGGGCAGTCTGCGACTTTCCTGTGCCGCATCAGGAAGGACCTCCAGCATGTATGCGATGGGATGGTTCCGACAAGCTCCGGGAAATGAGCGCGAGTTTGTTGCGGGAATTGGCTGGGAGAATAACACGCCCTATTATGCTCGGTCCGTAGAGGGGAGGTTCACTATCAGCCGAGATAATGTAAAAAACACCGTATTCCTCCAAATGAATCGGTTGAAACCAGAGGACGCAGCGGTCTACTTTTGCGCCGCGCAAATCGGCATAAGCGGTACATTGGGGGATTACTGGGGTCAAGGCACACAGGTAACCGTCTCTAGTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA(SEQ IDNO:37)
实施例9.检测人源化抗体对抗原PD-1的亲和力
参考实施例5的实验步骤,检测人源化抗体对抗原PD-1的亲和力。试验结果见图3,结果显示huVHH3-1-Fc、huVHH3-2-Fc、huVHH3-3-Fc、huVHH3-4-Fc、huVHH3-5-Fc、huVHH3-6-Fc结合PD-1蛋白的EC50分别为29.25ng/ml、40.50ng/ml、46.96ng/ml、13.31ng/ml、47.61ng/ml和45.97ng/ml。实验结果说明,六种人源化抗体在人源化以后亲和力与母本抗体类似或者更好。
实施例10.检测人源化抗体在体外阻断PD-1/PD-L1信号通路的细胞活性
参考实施例6的实验步骤,检测人源化抗体在体外阻断PD-1/PD-L1信号通路的细胞活性。试验结果见图4。实验结果说明,六种人源化抗体在人源化以后在体外细胞水平表现出与母本抗体类似或者更好的生物学效果。
实施例11.六种人源化抗体的亲和力测定
本次实验使用Fortebio公司的Octet RED 96e型分子相互作用分析仪。使用Protein A探针(Fortebio)先分别捕获候选抗体分子(浓度为5μg/mL)。再将探针分别浸入30nM或120nM的人PD-1抗原溶液中,使候选抗体分子与抗原结合时间为180s。然后将探针浸入缓冲液中,解离时间为1400s,测量结合与解离信号。使用软件Octet data analysissoftware拟合结合解离曲线来确定亲和力KD值。
实验结果见表4。由表4的结果可以看出六种PD-1单域重组抗体都可以以较高的亲和力结合抗原并且对抗原结合的亲和力差别不大。
表4.六种人源化PD-1重组抗体与抗原的结合情况
抗原 | kon(1/Ms) | kdis(1/s) | KD(M) |
LL-VHH01-Fc | 7.94E+04 | 2.01E-04 | 2.53E-09 |
hu-VHH3-1-Fc | 8.40E+04 | 2.40E-04 | 2.86E-09 |
hu-VHH3-2-Fc | 7.91E+04 | 2.31E-04 | 2.92E-09 |
hu-VHH3-3-Fc | 8.91E+04 | 2.22E-04 | 2.49E-09 |
hu-VHH3-4-Fc | 7.45E+04 | 3.08E-04 | 4.13E-09 |
hu-VHH3-5-Fc | 8.46E+04 | 2.25E-04 | 2.66E-09 |
hu-VHH3-6-Fc | 9.42E+04 | 2.67E-04 | 2.83E-09 |
实施例12.人源化PD-1单域抗体的体内药效学评估
取50只6-8周龄雌性hPD-1C57(PD-1基因人源化)人源化小鼠(百奥塞图),将MC38WT细胞以1×106个/0.1mL浓度接种小鼠的右侧皮下,待肿瘤生长到约100mm3时将小鼠按肿瘤体积随机分组,每组5只,共8组,分别为:
组1KLH IgG4(0.3mg/kg)阴性对照组、组2LL-Pos(0.3mg/kg)阳性对照组、组3huVHH3-1-Fc(0.3mg/kg)治疗组、组4huVHH3-3-Fc(0.3mg/kg)治疗组、组5huVHH3-5-Fc(0.3mg/kg)治疗组。
所有组的给药途径均为腹腔注射,给药剂量为0.3mg/kg,给药浓度为0.03mg/ml。每周给药2次,连续给药5次,末次给药3天后结束实验。每周测量肿瘤体积及体重2次,记录小鼠体重和肿瘤体积。实验结束时将小鼠安乐死,计算相对肿瘤抑制率TGI%=(1-(Ti-T0)/(Vi-V0))×100%。(Ti:治疗组或阳性对照组在给药第i天的肿瘤体积均值;T0:治疗组或阳性对照组在给药第0天的肿瘤体积均值;Vi:阴性对照组在给药第i天的肿瘤体积均值;V0:阴性对照组在给药第0天的肿瘤体积均值)。
如图5所示,在小鼠接种肿瘤细胞后第28天,阴性对照组平均肿瘤体积为1375±115mm3,阳性对照组平均肿瘤体积为494±267mm3,与阴性对照组相比,相对肿瘤抑制率为69.10%;组3、组4、组5治疗组平均肿瘤体积分别为823±85mm3、483±197mm3和320±229mm3,与阴性对照相比,相对肿瘤抑制率分别为43.30%、70.00%和82.75%,表明上述人源化抗PD-1单域抗体能够体内抑制hPD-1人源化小鼠MC38-WT细胞皮下移植瘤的生长,且类似或者明显优于阳性对照抗体。
实施例13.代表性人源化抗体的化学稳定性和血清稳定性检测
将候选分子置换到pH 5.5的缓冲液中(20mM醋酸-醋酸钠,220mM海藻糖,0.02%聚山梨酯80),控制样品浓度在10mg/ml左右,分装一定样品体积(200μl/管)放置40℃恒温箱,考察0周、2周和4周稳定性,根据时间点送样检测SEC-HPLC纯度和结合ELISA活性。检测结果显示3个候选分子具有良好的化学稳定性,单体纯度和结合ELISA活性未见明显下降,详见表5。
表5.三种代表性人源化抗体的化学稳定性检测
将候选分子添加到人血清中,控制样品浓度在1mg/ml左右,分装一定样品体积(200μl/管)放置37℃恒温箱,考察0周、2周和4周稳定性,根据时间点送样检测结合ELISA活性。检测结果显示3个候选分子具有良好的血清稳定性,结合ELISA活性未见明显下降,详见表6。
表6.三种代表性人源化抗体的血清稳定性检测
以上结果说明,本发明中PD-1人源化单域抗体具有很好的化学稳定性和血清稳定性,可以进行后续开发。
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。
序列表
<110> 立凌生物制药(苏州)有限公司
<120> 靶向PD-1的单域抗体及其衍生物和用途
<130> P2021-0470
<150> CN2021100481961
<151> 2021-01-14
<160> 37
<170> PatentIn version 3.5
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cagttgcagc tcgtggagtc gggaggaggg ctggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg cacctccagt atgtatgcca tgggctggtt ccgccagtct 120
ccagggaacg agcgcgagtt tgtagcgggg attggctggg agaataatac cccatactat 180
gcacgctccg tggagggccg attcaccatc tccagagaca acgtcaagaa cacggtcttt 240
ctacaaatga acagactgaa acctgaggac gcggccgttt atttttgtgc agcccaaatc 300
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35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
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Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
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cagttgcagc tcgtggagtc gggaggaggg ctggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg cacctccagt atgtatgcca tgggctggtt ccgccagtct 120
ccagggaacg agcgcgagtt tgtagcgggg attggctggg agaataatac cccatactat 180
gcacgctccg tggagggccg attcaccatc tccagagaca acgtcaagaa cacggtcttt 240
ctacaaatga acagactgaa acctgaggac gcggccgttt atttttgtgc agcccaaatc 300
ggaatatccg gtacattggg ggactactgg ggccagggga cccaggtcac cgtctcctca 360
gagtccaaat atggtccccc atgcccacca tgcccagcac ctgagttcct ggggggacca 420
tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 480
gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 540
gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 600
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 660
tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 720
gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 780
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 840
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 900
gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 960
gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1020
aagagcctct ccctgtctct gggtaaa 1047
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Ser Met Tyr
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Ala Met Gly Trp Phe Arg Gln Ser Pro Gly Asn Glu Arg Glu Phe Val
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Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
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Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
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Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
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His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
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Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
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Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
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Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
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Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
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His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
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Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
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Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser
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Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
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Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala
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Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
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Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg
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Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
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Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
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Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
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Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
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His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Ser Met Tyr
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Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val
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Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
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Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Tyr
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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
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Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
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Gly Thr Gln Val Thr Val Ser Ser
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<210> 15
<211> 360
<212> DNA
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<220>
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<400> 15
gaggtgcagc ttgttgaaag tggtggaggt cttgttcaac cagggggctc cctcagactg 60
tcttgtgcgg cgagcgggcg gacatcctct atgtatgcga tgggttggtt ccgacaggcc 120
cccggtaaag gacgggagtt cgtagctggc atcggttggg aaaacaatac cccttattac 180
gcccggtctg ttgaaggtcg atttactata agtcgggaca atgtgaaaaa tactgtctat 240
ctccaaatga actctctgcg ggccgaagat acagcggtgt actattgtgc cgcccaaatt 300
ggaatcagcg gaacattggg tgattattgg ggccaaggta cgcaagttac agtctcctca 360
<210> 16
<211> 120
<212> PRT
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<220>
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<400> 16
Glu Val Gln Leu Val 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 Ser Ser Met Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 17
<211> 360
<212> DNA
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<220>
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<400> 17
gaagtccaac tggtcgaaag cggcggcggt ctcgtccaac ctggaggctc tcttaggttg 60
tcatgtgccg cctcaggcag aacatccagc atgtacgcaa tgggttggtt cagacaggct 120
ccggggaacg agcgagaatt cgtcgcggga ataggatggg agaacaacac cccatactac 180
gcacgcagtg tggaaggccg attcactatt agtcgggata atgttaaaaa cacggtctac 240
cttcaaatga actcccttcg cgcagaggat actgcagttt attattgcgc ggcccaaata 300
ggtataagtg gaacactcgg ggactactgg ggccagggaa cacaggtaac cgtatcatca 360
<210> 18
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> 合成的多肽
<400> 18
Glu Val Gln Leu Val 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 Ser Ser Met Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Phe
65 70 75 80
Leu Gln Met Asn Arg Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 19
<211> 360
<212> DNA
<213> 人工序列
<220>
<223> 合成的多核苷酸
<400> 19
gaggtccagt tggtagaaag tggtggtggg ttggtgcaac ccggtggctc attgaggctg 60
tcttgtgctg cgagtggcag gacatcctct atgtatgcga tgggatggtt ccgacaagct 120
ccaggaaacg agcgcgagtt cgtagccgga attggttggg aaaacaatac gccctattat 180
gcacggtctg tcgaggggag gttcactatc tcacgcgaca acgtcaagaa cacagtgttt 240
cttcagatga accgactccg ggcggaggat acggccgtat attattgcgc agcgcaaatc 300
ggtatatccg gcactcttgg tgactattgg ggccagggta cacaagtgac agtctcttca 360
<210> 20
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> 合成的多肽
<400> 20
Glu Val Gln Leu Val 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 Ser Ser Met Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Phe
65 70 75 80
Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 21
<211> 360
<212> DNA
<213> 人工序列
<220>
<223> 合成的多核苷酸
<400> 21
gaagtgcaac tcgtggagag cgggggcgga cttgtccaac cgggagggag tttgagactc 60
tcatgcgccg cctctggtag aactagcagc atgtacgcta tgggatggtt caggcaggct 120
ccagggaacg aacgagaatt cgttgcaggc ataggatggg aaaacaacac cccatattac 180
gctcggtccg tggaaggacg atttactata agccgggaca atgtaaaaaa tactgtcttt 240
ctccagatga ataggctcaa gccggaggat acagcagttt attattgcgc tgctcaaatt 300
gggattagcg ggaccctggg tgactattgg gggcagggaa cgcaagtgac tgtcagttct 360
<210> 22
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> 合成的多肽
<400> 22
Glu Leu Gln Leu Val 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 Ser Ser Met Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Phe
65 70 75 80
Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 23
<211> 360
<212> DNA
<213> 人工序列
<220>
<223> 合成多核苷酸
<400> 23
gagttgcaac tggtggaaag tggtggcggg ttggttcagg caggcggttc ccttcgcctc 60
tcctgtgcgg cgagtggacg cacatcatcc atgtacgcaa tggggtggtt tcgacaagcc 120
cccggaaacg aacgcgaatt tgttgctggg attggatggg aaaacaatac gccgtactat 180
gcccggagcg tagaaggacg attcaccatt tccagggaca acgtcaaaaa cacggtcttc 240
ttgcaaatga accgcttgaa accagaggat accgcagtat actattgtgc tgcccagatc 300
ggcatatcag gcacactggg cgactattgg ggccaaggga cccaggtcac tgtatccagc 360
<210> 24
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> 合成的多肽
<400> 24
Glu Leu Gln Leu Val 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 Ser Ser Met Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Phe
65 70 75 80
Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Ala Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 25
<211> 360
<212> DNA
<213> 人工序列
<220>
<223> 合成的多核苷酸
<400> 25
gaactgcaac tcgtagaatc tgggggtggc ttggtccagg ccgggggcag tctgcgactt 60
tcctgtgccg catcaggaag gacctccagc atgtatgcga tgggatggtt ccgacaagct 120
ccgggaaatg agcgcgagtt tgttgcggga attggctggg agaataacac gccctattat 180
gctcggtccg tagaggggag gttcactatc agccgagata atgtaaaaaa caccgtattc 240
ctccaaatga atcggttgaa accagaggac gcagcggtct acttttgcgc cgcgcaaatc 300
ggcataagcg gtacattggg ggattactgg ggtcaaggca cacaggtaac cgtctctagt 360
<210> 26
<211> 349
<212> PRT
<213> 人工序列
<220>
<223> 合成的多肽
<400> 26
Glu Val Gln Leu Val 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 Ser Ser Met Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys
115 120 125
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
130 135 140
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
145 150 155 160
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
165 170 175
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
180 185 190
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
210 215 220
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 27
<211> 1047
<212> DNA
<213> 人工序列
<220>
<223> 合成的多核苷酸
<400> 27
gaggtgcagc ttgttgaaag tggtggaggt cttgttcaac cagggggctc cctcagactg 60
tcttgtgcgg cgagcgggcg gacatcctct atgtatgcga tgggttggtt ccgacaggcc 120
cccggtaaag gacgggagtt cgtagctggc atcggttggg aaaacaatac cccttattac 180
gcccggtctg ttgaaggtcg atttactata agtcgggaca atgtgaaaaa tactgtctat 240
ctccaaatga actctctgcg ggccgaagat acagcggtgt actattgtgc cgcccaaatt 300
ggaatcagcg gaacattggg tgattattgg ggccaaggta cgcaagttac agtctcctca 360
gagtccaaat atggtccccc atgcccacca tgcccagcac ctgagttcct ggggggacca 420
tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 480
gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 540
gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 600
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 660
tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 720
gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 780
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 840
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 900
gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 960
gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1020
aagagcctct ccctgtctct gggtaaa 1047
<210> 28
<211> 349
<212> PRT
<213> 人工序列
<220>
<223> 合成的多肽
<400> 28
Glu Val Gln Leu Val 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 Ser Ser Met Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys
115 120 125
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
130 135 140
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
145 150 155 160
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
165 170 175
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
180 185 190
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
210 215 220
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 29
<211> 1047
<212> DNA
<213> 人工序列
<220>
<223> 合成的多核苷酸
<400> 29
gaagtccaac tggtcgaaag cggcggcggt ctcgtccaac ctggaggctc tcttaggttg 60
tcatgtgccg cctcaggcag aacatccagc atgtacgcaa tgggttggtt cagacaggct 120
ccggggaacg agcgagaatt cgtcgcggga ataggatggg agaacaacac cccatactac 180
gcacgcagtg tggaaggccg attcactatt agtcgggata atgttaaaaa cacggtctac 240
cttcaaatga actcccttcg cgcagaggat actgcagttt attattgcgc ggcccaaata 300
ggtataagtg gaacactcgg ggactactgg ggccagggaa cacaggtaac cgtatcatca 360
gagtccaaat atggtccccc atgcccacca tgcccagcac ctgagttcct ggggggacca 420
tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 480
gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 540
gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 600
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 660
tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 720
gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 780
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 840
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 900
gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 960
gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1020
aagagcctct ccctgtctct gggtaaa 1047
<210> 30
<211> 349
<212> PRT
<213> 人工序列
<220>
<223> 合成的多肽
<400> 30
Glu Val Gln Leu Val 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 Ser Ser Met Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Phe
65 70 75 80
Leu Gln Met Asn Arg Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys
115 120 125
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
130 135 140
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
145 150 155 160
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
165 170 175
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
180 185 190
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
210 215 220
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 31
<211> 1047
<212> DNA
<213> 人工序列
<220>
<223> 合成的多核苷酸
<400> 31
gaggtccagt tggtagaaag tggtggtggg ttggtgcaac ccggtggctc attgaggctg 60
tcttgtgctg cgagtggcag gacatcctct atgtatgcga tgggatggtt ccgacaagct 120
ccaggaaacg agcgcgagtt cgtagccgga attggttggg aaaacaatac gccctattat 180
gcacggtctg tcgaggggag gttcactatc tcacgcgaca acgtcaagaa cacagtgttt 240
cttcagatga accgactccg ggcggaggat acggccgtat attattgcgc agcgcaaatc 300
ggtatatccg gcactcttgg tgactattgg ggccagggta cacaagtgac agtctcttca 360
gagtccaaat atggtccccc atgcccacca tgcccagcac ctgagttcct ggggggacca 420
tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 480
gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 540
gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 600
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 660
tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 720
gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 780
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 840
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 900
gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 960
gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1020
aagagcctct ccctgtctct gggtaaa 1047
<210> 32
<211> 349
<212> PRT
<213> 人工序列
<220>
<223> 合成的多肽
<400> 32
Glu Val Gln Leu Val 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 Ser Ser Met Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Phe
65 70 75 80
Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys
115 120 125
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
130 135 140
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
145 150 155 160
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
165 170 175
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
180 185 190
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
210 215 220
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 33
<211> 1047
<212> DNA
<213> 人工序列
<220>
<223> 合成的多核苷酸
<400> 33
gaagtgcaac tcgtggagag cgggggcgga cttgtccaac cgggagggag tttgagactc 60
tcatgcgccg cctctggtag aactagcagc atgtacgcta tgggatggtt caggcaggct 120
ccagggaacg aacgagaatt cgttgcaggc ataggatggg aaaacaacac cccatattac 180
gctcggtccg tggaaggacg atttactata agccgggaca atgtaaaaaa tactgtcttt 240
ctccagatga ataggctcaa gccggaggat acagcagttt attattgcgc tgctcaaatt 300
gggattagcg ggaccctggg tgactattgg gggcagggaa cgcaagtgac tgtcagttct 360
gagtccaaat atggtccccc atgcccacca tgcccagcac ctgagttcct ggggggacca 420
tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 480
gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 540
gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 600
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 660
tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 720
gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 780
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 840
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 900
gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 960
gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1020
aagagcctct ccctgtctct gggtaaa 1047
<210> 34
<211> 349
<212> PRT
<213> 人工序列
<220>
<223> 合成的多肽
<400> 34
Glu Leu Gln Leu Val 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 Ser Ser Met Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Phe
65 70 75 80
Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys
115 120 125
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
130 135 140
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
145 150 155 160
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
165 170 175
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
180 185 190
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
210 215 220
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 35
<211> 1047
<212> DNA
<213> 人工序列
<220>
<223> 合成的多核苷酸
<400> 35
gagttgcaac tggtggaaag tggtggcggg ttggttcagg caggcggttc ccttcgcctc 60
tcctgtgcgg cgagtggacg cacatcatcc atgtacgcaa tggggtggtt tcgacaagcc 120
cccggaaacg aacgcgaatt tgttgctggg attggatggg aaaacaatac gccgtactat 180
gcccggagcg tagaaggacg attcaccatt tccagggaca acgtcaaaaa cacggtcttc 240
ttgcaaatga accgcttgaa accagaggat accgcagtat actattgtgc tgcccagatc 300
ggcatatcag gcacactggg cgactattgg ggccaaggga cccaggtcac tgtatccagc 360
gagtccaaat atggtccccc atgcccacca tgcccagcac ctgagttcct ggggggacca 420
tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 480
gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 540
gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 600
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 660
tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 720
gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 780
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 840
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 900
gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 960
gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1020
aagagcctct ccctgtctct gggtaaa 1047
<210> 36
<211> 349
<212> PRT
<213> 人工序列
<220>
<223> 合成的多肽
<400> 36
Glu Leu Gln Leu Val 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 Ser Ser Met Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Gly Trp Glu Asn Asn Thr Pro Tyr Tyr Ala Arg Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Phe
65 70 75 80
Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Ala Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gln Ile Gly Ile Ser Gly Thr Leu Gly Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys
115 120 125
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
130 135 140
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
145 150 155 160
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
165 170 175
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
180 185 190
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
210 215 220
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 37
<211> 1047
<212> DNA
<213> 人工序列
<220>
<223> 合成的多核苷酸
<400> 37
gaactgcaac tcgtagaatc tgggggtggc ttggtccagg ccgggggcag tctgcgactt 60
tcctgtgccg catcaggaag gacctccagc atgtatgcga tgggatggtt ccgacaagct 120
ccgggaaatg agcgcgagtt tgttgcggga attggctggg agaataacac gccctattat 180
gctcggtccg tagaggggag gttcactatc agccgagata atgtaaaaaa caccgtattc 240
ctccaaatga atcggttgaa accagaggac gcagcggtct acttttgcgc cgcgcaaatc 300
ggcataagcg gtacattggg ggattactgg ggtcaaggca cacaggtaac cgtctctagt 360
gagtccaaat atggtccccc atgcccacca tgcccagcac ctgagttcct ggggggacca 420
tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 480
gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 540
gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 600
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 660
tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 720
gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 780
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 840
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 900
gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 960
gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1020
aagagcctct ccctgtctct gggtaaa 1047
Claims (13)
1.一种靶向PD-1的单域抗体,所述单域抗体包括SEQ ID NO: 2所示的CDR1、SEQ IDNO: 3所示的CDR2和SEQ ID NO: 4所示的CDR3。
2.一种人源化的靶向PD-1的单域抗体,其特征在于,以权利要求1所述的单域抗体为基础对框架区FR1、FR2、FR3和FR4进行人源化。
3.一种靶向PD-1的抗体,所述抗体包括一个或多个如权利要求1所述的靶向PD-1的单域抗体或如权利要求2所述的人源化的靶向PD-1的单域抗体。
4.一种双特异性抗体,所述双特异性抗体包括第一抗体和第二抗体,所述第一抗体包括权利要求1所述的靶向PD-1的单域抗体、或权利要求2所述的人源化的靶向PD-1的单域抗体、或权利要求3所述的靶向PD-1的抗体。
5.一种融合蛋白,所述融合蛋白包括权利要求1所述的靶向PD-1的单域抗体、权利要求2所述的人源化的靶向PD-1的单域抗体、或权利要求3所述的靶向PD-1的抗体,任选的接头序列以及免疫球蛋白的Fc片段。
6.一种核酸分子,所述核酸分子编码权利要求1所述的靶向PD-1的单域抗体、权利要求2所述的人源化的靶向PD-1的单域抗体、权利要求3所述的靶向PD-1的抗体、权利要求4所述的双特异性抗体或权利要求5所述的融合蛋白。
7.一种表达载体,所述表达载体包含权利要求6所述的核酸分子。
8.一种宿主细胞,所述宿主细胞包含权利要求7所述的表达载体,或者其基因组上整合有权利要求6所述的核酸分子。
9. 一种制备权利要求1所述的靶向PD-1的单域抗体、权利要求2所述的人源化的靶向PD-1的单域抗体、权利要求3所述的靶向PD-1的抗体、权利要求4所述的双特异性抗体、或权利要求5所述的融合蛋白的方法,所述方法包括以下步骤:
1) 在适合的条件下,培养如权利要求8所述的宿主细胞,从而获得含有所述的靶向PD-1的单域抗体的VHH链、靶向PD-1的抗体的重链可变区、靶向PD-1的单域抗体、人源化的靶向PD-1的单域抗体的VHH链、靶向PD-1的抗体、双特异性抗体或融合蛋白的培养物;以及
2) 任选地,从所述培养物中分离或回收所述的靶向PD-1的单域抗体、人源化的靶向PD-1的单域抗体、靶向PD-1的单域抗体、双特异性抗体或融合蛋白。
10. 一种免疫偶联物,所述免疫偶联物含有:
1) 如权利要求1所述的靶向PD-1的单域抗体、权利要求2所述的人源化的靶向PD-1的单域抗体、权利要求3所述的靶向PD-1的抗体、权利要求4所述的双特异性抗体或权利要求5所述的融合蛋白;和
2) 选自以下的偶联部分:可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
11.一种药物组合物,所述药物组合物包含治疗或诊断有效量的权利要求1所述的靶向PD-1的单域抗体、权利要求2所述的人源化的靶向PD-1的单域抗体、权利要求3所述的靶向PD-1的抗体、权利要求4所述的双特异性抗体、权利要求5所述的融合蛋白或权利要求10所述的免疫偶联物,和任选的药学上可接受的赋形剂。
12.权利要求1所述的靶向PD-1的单域抗体、权利要求2所述的人源化的靶向PD-1的单域抗体、权利要求3所述的靶向PD-1的抗体、权利要求4所述的双特异性抗体、权利要求5所述的融合蛋白或权利要求10所述的免疫偶联物的用途,用于制备以下试剂:
1) 检测PD-1的试剂;
2) 阻断PD-1与PD-L1结合的试剂;
3) ***的药物。
13.一种试剂盒,所述试剂盒中包括:
1) 权利要求1所述的靶向PD-1的单域抗体、权利要求2所述的人源化的靶向PD-1的单域抗体、权利要求3所述的靶向PD-1的抗体、权利要求4所述的双特异性抗体、权利要求5所述的融合蛋白、权利要求10所述的免疫偶联物或权利要求11所述的药物组合物;
2) 容器;和
3) 任选的使用说明书。
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