CN115028726B - 一种抗pd-1纳米抗体及其应用 - Google Patents
一种抗pd-1纳米抗体及其应用 Download PDFInfo
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Abstract
本发明提供了一种新型的抗PD‑1纳米抗体,其为纳米抗体VHH链与包含经修饰铰链区的IgG4‑Fc的融合蛋白。该纳米抗体在表达纯化后无絮状物、均一性好,对抗原PD‑1的结合亲和力高,体内抑瘤效果好,适用于工业化生产和商品化应用。本发明还提供了该纳米抗体的应用。
Description
技术领域
本发明属于生物制药技术领域,具体涉及针对程序性死亡受体1(PD-1)的纳米抗体及其应用。
背景技术
程序性死亡受体1(PD-1)属于免疫球蛋白超家族CD28/B7,是由288个氨基酸组成的Ⅰ型跨膜糖蛋白,为免疫抑制性受体。PD-1在结构上由胞外结构域、疏水性跨区和胞质区组成,其中,胞外结构域包含一个IgV(免疫球蛋白可变区)结构域,与细胞毒性T淋巴细胞抗原4(CTLA-4)等其他共刺激分子具有22-33%的同源性,同时含有4个能被糖基化的位点;胞质尾部含有氨基端和羧基端两个酪氨酸残基,分别构成一个免疫受体酪氨酸抑制基序(ITIM)和一个免疫受体酪氨酸转换基序(ITSM)。
PD-1的主要配体为PD-L1,其又称B7-H1,是一种由人CD274基因编码的Ⅰ型跨膜糖蛋白。PD-L1含IgV样区、IgC(免疫球蛋白恒定区)样区、跨膜区和细胞质尾区,其中细胞质尾区与细胞内的信号转导相关;IgV区和IgC区则参与细胞间的信号转导。
PD-1与PD-L1结合时,通过发生磷脂酰肌醇-3-激酶的磷酸化、蛋白激酶B的进一步激活、刺激性T细胞信号通路的活化、葡萄糖代谢以及干扰素的分泌等,导致T细胞活化的下游信号受阻,进而有效抑制T细胞的转录,最终抑制T细胞的免疫功能,在免疫应答的负性调控方面发挥着重要作用。因此,阻断PD-1/PD-L1信号通路可使T细胞活化上调,激活内源性抗肿瘤免疫反应,从而发挥对肿瘤的治疗作用。
(/>帕博利珠单抗)为默沙东公司研制的一款全球领先的抗PD-1单抗,分别于2014年9月、2015年7月及2018年7月在美国、欧洲和中国上市。这款重磅肿瘤免疫疗法在多种癌症中展现出了喜人的疗效,可用于治疗黑色素瘤、结直肠癌、头颈癌、非小细胞肺癌、经典霍奇金淋巴瘤、膀胱癌、胃癌等,目前获批的适应症达13个。2021年Keytruda全球销售额高达171.8亿美元,在一众抗肿瘤单抗药物中成为绝对的TOP1产品。但是,Keytruda虽然取得了商业上的巨大成功,然而其为传统的单抗,相对纳米抗体在稳定性、生产成本等方面存在一定的劣势。
纳米抗体(nanobody,Nb),即重链单域抗体VHH(variable domain of heavychain of heavy-chain antibody),直径2.5nm,长4nm,是自然存在的可以和抗原结合的最小片段。纳米抗体既能在哺乳动物中表达,又可在大肠杆菌表达系统中表达,表达的抗体具有较低的免疫原性,更容易廉价批量生产,在更宽的温度和pH范围内稳定性更高,相比传统抗体具有一定的优势。
CN107814845A为申请人在先专利,公开了一种抗PD-1纳米抗体,该文件描述了:利用人源的PD-1抗原蛋白免疫骆驼,获得免疫纳米抗体基因库;将PD-1蛋白分子偶联在酶标板上,展示PD-1蛋白的空间结构,然后以此形式的抗原利用噬菌体展示技术筛选获得的免疫纳米抗体基因库,从而获得PD-1特异性的纳米抗体基因;再将此基因转至大肠杆菌中,获得了能在大肠杆菌中高效表达的、且特异性高的纳米抗体株。此外,CN107814845A还公开了通过对骆驼源的纳米抗体进行人源化改造而获得的人源化的抗PD-1纳米抗体。
但是,CN107814845A中在载体构建时,将纳米抗体VHH序列合成到pFUSE-hIgG1-Fc2载体上,提取pFUSE-Nb-hIgG1-Fc2质粒后,转染至真核细胞HEK293中进行表达及纯化,得到了VHH-IgG1-Fc抗体。但是,该抗体进行纯化后,产物样品经外观检测发现有明显絮状物;CE-SDS检测显示出明显的分裂峰,表明该抗体均一性相对较差,申请人认为其难以达到作为药物开发的候选分子的标准,须按照药物开发的思维和方法对其进行改进,以满足药物开发及商业化生产的要求。
发明内容
本发明的目的在于,基于CN107814845A中公开的纳米抗体序列,提供改进的抗PD-1纳米抗体,该纳米抗体保留对抗原PD-1的高亲和力但均一性得到明显改善,并具有良好药物疗效,药物疗效得到显著提升,符合将其作为药物开发候选分子的要求,并适于工业化生产,且具有临床应用潜力。
本发明的技术方案如下:
一方面,本发明提供一种抗PD-1纳米抗体,其由VHH链与包含经修饰铰链区的IgG4-Fc组成,其中:
(i)所述抗PD-1纳米抗体VHH链包含SEQ ID NO.2所示的CDR1、SEQ ID NO.3所示的CDR2、SEQ ID NO.4所示的CDR3及SEQ ID NO.28所示的CDR1和CDR2之间的框架区FR2;并且
(ii)所述抗PD-1纳米抗体中的经修饰铰链区包含SEQ ID NO:9所示氨基酸序列,或者由连接肽与IgG4-Fc中的铰链区序列组成,所述连接肽包含SEQ ID NO.12、SEQ IDNO.21或SEQ ID NO.24所示氨基酸序列。
在本发明中,术语“抗PD-1纳米抗体”、“抗PD-1抗体”和“融合蛋白”根据上下文可互换使用。
本发明的抗PD-1纳米抗体VHH链包含的CDR1、CDR2和CDR3源自SEQ ID NO.1。
SEQ ID NO.1:
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSS
CN107814845A公开了包含SEQ ID NO:1所示氨基酸序列的VHH链的参照纳米抗体(VHH-IgG1-Fc),本领域技术人员可以常规地确定其包含的VHH链CDR1、CDR2和CDR3区段。以本领域已知方法划分得到的VHH链CDR1、CDR2和CDR3组合均被涵盖在本发明的范围内。
根据本发明的具体实施方式,在本发明提供的抗PD-1纳米抗体中,所述VHH链CDR1、CDR2和CDR3的氨基酸序列以及框架区FR2的氨基酸序列分别如下所示:
SEQ ID NO.2:
GSTYLRFSMG
SEQ ID NO.3:
IGGDGRT
SEQ ID NO.4:
AAAVLLDGSFSLLAPLVPYKYDY
SEQ ID NO.28:
WFRQVPGKEREGVAA
根据本发明的具体实施方式,在本发明提供的抗PD-1纳米抗体中,所述经修饰铰链区的氨基酸序列或者所述连接肽的氨基酸序列分别如下所示:
经修饰铰链区的氨基酸序列如SEQ ID NO.9所示:
ERKSSVECPPCP
修饰前IgG4-Fc中的铰链区序列如SEQ ID NO.6所示:
ESKYGPPCPPCP
连接肽氨基酸序列如SEQ ID NO.12、SEQ ID NO.21或SEQ ID NO.24所示:
SEQ ID NO.12:
EPKIPQPQPKPQPQPQPQPKPQPKPEPE
SEQ ID NO.21:
EPKIPQPQPK
SEQ ID NO.24:
EPKIPQPQPKPQPKPEPE
所述连接肽连接至SEQ ID NO.6所示的修饰前IgG4-Fc铰链区的N端。
优选地,本发明提供的抗PD-1纳米抗体为抗人PD-1纳米抗体。
进一步地,在本发明提供的抗PD-1纳米抗体中,所述VHH链包含上述CDR1、CDR2和CDR3以及其间的4个框架区(framework region,FR),各个区的排列方式为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。优选地,在本发明提供的抗PD-1纳米抗体中,所述VHH链包含SEQ IDNO.27所示的FR1、SEQ ID NO.29所示的FR3和SEQ ID NO.30所示的FR4。
SEQ ID NO.27:
QVQLQESGGGLVQPGGSLRLSCAAS
SEQ ID NO.29:
SYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYC
SEQ ID NO.30:
WGQGTLVTVSS
根据本发明的具体实施方式,在本发明提供的抗PD-1纳米抗体中,所述VHH链包含SEQ ID NO.7所示氨基酸序列。
SEQ ID NO.7:
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSS
优选地,本发明提供的抗PD-1纳米抗体包含SEQ ID NO.10、SEQ ID NO.13、SEQ IDNO.22或SEQ ID NO.25所示氨基酸序列。
SEQ ID NO.10:
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCP
SEQ ID NO.13:
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCP
SEQ ID NO.22:
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCP
SEQ ID NO.25:
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCP
进一步地,根据本发明的具体实施方式,本发明提供的抗PD-1纳米抗体由VHH链与包含经修饰铰链区的IgG4-Fc组成,所述纳米抗体氨基酸全长序列如SEQ ID NO.11、SEQ IDNO.14、SEQ ID NO.23或SEQ ID NO.26所示。
SEQ ID NO.11:
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO.14:
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO.23:
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO.26:
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
另一方面,本发明提供一种核酸分子,所述核酸分子包含核苷酸序列,所述核苷酸序列编码本发明提供的抗PD-1纳米抗体中包含的氨基酸序列。所述氨基酸序列可以是上文提供的任一序列。本发明所述的核苷酸序列可以为DNA序列或RNA序列。所述核酸分子可以是DNA分子或RNA分子,可以是单链或双链的。
再一方面,本发明提供一种载体,所述载体含有本发明提供的核酸分子。优选地,所述载体可以为表达载体。根据本发明的具体实施方式,所述载体为真核细胞表达载体或原核细胞表达载体。
还一方面,本发明提供一种宿主细胞,所述宿主细胞含有本发明提供的载体,例如被所述载体转化或转染,或所述宿主细胞在基因组中整合有本发明提供的核酸分子。根据本发明的具体实施方式,所述宿主细胞为真核细胞,例如真菌、昆虫、植物或动物细胞,或为原核细胞,例如细菌细胞。
另一方面,本发明提供一种组合物,所述组合物包含本发明提供的抗PD-1纳米抗体、核酸分子、载体和/或宿主细胞。
再一方面,本发明提供所述抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物在制备用于治疗PD-1或PD-L1相关疾病的药物中的用途。优选地,所述疾病为肿瘤或癌症,或感染性疾病。进一步优选地,所述疾病为肺癌、胃癌、肝癌、黑色素瘤、宫颈癌、结直肠癌、膀胱癌、乳腺癌、白血病、淋巴瘤、肾细胞癌、盲肠癌、胰腺癌、胆管癌、头颈癌、梅克尔细胞癌、卵巢癌、鼻咽癌、胶质瘤、食管癌、骨癌或前列腺癌。优选地,所述疾病为结直肠癌。
因此相应地,本发明还提供一种治疗PD-1或PD-L1相关疾病的方法,所述方法包括给有此需要的受试者施用本发明提供的抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物。优选地,所述疾病为肿瘤(包括恶性肿瘤或癌症)或感染性疾病。进一步优选地,所述疾病为肺癌、胃癌、肝癌、黑色素瘤、宫颈癌、结直肠癌、膀胱癌、乳腺癌、白血病、淋巴瘤、肾细胞癌、盲肠癌、胰腺癌、胆管癌、头颈癌、梅克尔细胞癌、卵巢癌、鼻咽癌、胶质瘤、食管癌、骨癌或前列腺癌。所述受试者是脊椎动物,优选是哺乳动物,例如人、家畜和农用动物以及动物园、运动或宠物动物,比如绵羊、狗、马、猫、牛、大鼠、猪、猕猴。根据本发明的具体实施方式,所述受试者是人。
还一方面,本发明提供所述抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物在制备PD-1检测试剂中的用途。优选地,所述检测试剂为基于流式细胞术或酶联免疫吸附法的检测试剂。
因此相应地,本发明还提供一种PD-1检测试剂,所述PD-1检测试剂包含本发明提供的抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物。优选地,所述检测试剂为基于流式细胞术或酶联免疫吸附法的检测试剂。
本发明还提供一种检测来自受试者的样品中PD-1的方法,所述方法包括使本发明提供的抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物与所述样品相接触。
又一方面,本发明提供一种试剂盒,所述试剂盒包含本发明提供的抗PD-1纳米抗体、核酸分子、载体、宿主细胞和/或组合物。优选地,所述试剂盒为检测试剂盒,例如基于流式细胞术或酶联免疫吸附法的检测试剂盒。
另一方面,本发明提供一种产生所述抗PD-1纳米抗体的方法,所述方法包括以下步骤:
(a)培养本发明提供的宿主细胞,从而获得含所述抗PD-1纳米抗体的培养物;和,(b)从所述培养物中回收所述抗PD-1纳米抗体。
与现有技术相比,本发明提供了一种抗PD-1纳米抗体,所述纳米抗体为纳米抗体VHH链与包含经修饰铰链区的IgG4-Fc的融合蛋白,其是基于CN107814845A中公开的纳米抗体的改进纳米抗体。
考虑到CN107814845A中表达抗体时采用的真核细胞HEK293并非抗体工业化生产用菌株,采用的IgG1也非针对PD-1的常规抗体亚型(抗PD-1单抗治疗药物均为IgG4型,从而仅保留阻断功能,弱化ADCC类效应子功能),因此本发明的发明人将CN107814845A中纳米抗体VHH链序列合成到IgG4-Fc上并采用工业化使用的CHO细胞进行表达及纯化,结果发现,纯化后的VHH-IgG4-Fc(以下简称PR抗体)同样存在明显絮状物,经CE-SDS检测,也出现了明显的分裂峰,说明经IgG4-Fc优化后的抗体絮状物及均一性未得到明显改善,难以实现基于该VHH序列的抗体成药性研究、工业化生产及临床治疗应用。
鉴于此,本发明的发明人进一步从成药性研发角度对序列进行了优化,特别是对VHH链、铰链区和Fc区域中的多个氨基酸位点进行特定氨基酸置换,通过对所得蛋白的理化、生化性质(包括均一性、结合亲和力、抗原活性阻断作用以及体内抑瘤实验)检测,筛选得到了具有更优活性、更具应用价值的新型抗PD-1纳米抗体,特别是该纳米抗体在表达纯化后无絮状物、均一性好,对抗原PD-1的结合亲和力高,体内抑瘤效果显著提升,特别是疗效可与(/>帕博利珠单抗)匹敌,符合将其作为药物开发候选分子的要求,并适于工业化生产,且具有临床应用潜力。
附图说明
以下,结合附图来详细说明本发明的实施方案,其中:
图1:VHH-IgG1-Fc和VHH-IgG4-Fc(PR抗体)的CE-SDS检测结果。
图2:PR抗体与优化纳米抗体的序列比对结果。
图3:PR抗体与优化纳米抗体的CE-SDS检测结果。
图4:优化纳米抗体N7和N15的SEC检测结果。
图5:给药后肿瘤体积增长趋势图。
图6:试验结束后小鼠肿瘤重。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解,这些实施例仅用于说明本发明,其不以任何方式限制本发明的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的原料、试剂材料等,如无特殊说明,均为市售购买产品。
下述实施例中,采用以下程序:
(一)纳米抗体样品的制备与纯化:
a.合成编码纳米抗体序列的基因序列;
b.将合成的基因序列构建到pcDNA3.1载体上,转化至大肠杆菌DH5α菌株中,大摇并用Omega质粒大提试剂盒提取pcDNA3.1质粒,并过滤除菌;
c.将CHO-S细胞培养至5×106个/ml;
d.将步骤b得到的pcDNA3.1质粒与转染试剂PEI以1:3的比例在转染培养基ExpiCHOTM Expression Medium中混合均匀并静置20min,然后加入到步骤c的CHO-S细胞中,于37℃、6%CO2、115rpm培养5天;
e.培养产物离心,0.2μm滤膜过滤,得到离心上清;
f.采用ProA亲和层析法纯化离心上清,使用pH7.0的Tris体系缓冲液清洗掉杂蛋白和其他杂质;
g.使用24mM醋酸洗脱;
h.将得到的洗脱液用Tris Base回调pH至5.0,再用0.2μm滤膜过滤,得到待测样品。
(二)纳米抗体样品的外观及可见异物检测
样品纯化后收集于西林瓶,观测纳米抗体样品外观及可见异物情况。
(三)纳米抗体样品的CE-SDS检测:
a.溶液制备:分别配制稀释缓冲液,DTT,NEM,染色凝胶和脱色液,分子量标准品和变性剂;
b.样品处理:在样品中加入超纯水和还原试剂(蛋白230试剂盒提供的样品缓冲液,与1M DTT混合)稀释后变性;非还原条件下,样品加入120mM N乙基马来酰亚胺(NEM)溶液和非还原试剂(样品溶液与超纯水混合);将还原和非还原条件下的ladder(阶梯式标记物)和样品加热后加到芯片上;样品在充满聚合物溶液的微通道中进行电泳分离;
c.检测:基于一种荧光染料的激光诱导荧光,样品添加到聚合物溶液中,并以非共价结合到蛋白质-SDS胶束上;
d.结果分析:生物分析仪软件根据ladder(阶梯式标记物)自动确定大小,并计算电泳图中每个分离峰的百分比。
(四)纳米抗体样品的SEC检测
a.试剂制备:制备溶液A(100mM磷酸盐缓冲液、100mM硫酸钠,pH=2.8)、溶液B(100mM磷酸盐缓冲液、100mM硫酸钠,pH=7.0);
b.检测系统设置:运行“U3000SEC”项目和“3000”色谱系统,使用A溶液清洗A管路,B溶液清洗B管路,柱温设置为25℃,样品室温度设置为8℃,关闭清洗阀,用超纯水清洗系统后,平衡系统压;
c.分析样品:在Empower3上设置样品序列,分析6针标准品后,每个样品分析1针2mg/ml,所有样品分析完后再分析1针标准品,进样体积为5微升;然后加载并运行序列;
d.运行结束后,分析数据。
实施例1已知抗PD-1纳米抗体的检测
CN107814845A中公开了一种新的抗PD-1纳米抗体及其应用,该抗体的VHH序列如下:
SEQ ID NO.1(CDR1/CDR2/CDR3:SEQ ID NO.2/SEQ ID NO.3/SEQ ID NO.4,下划线并加粗示出):
按照上文“(一)纳米抗体样品的制备”所述程序制备VHH-IgG4-Fc(PR抗体),其中IgG4-Fc的氨基酸序列示于SEQ ID NO.5。
对CN107814845A中公开的在HEK293中表达的VHH-IgG1-Fc(参见CN107814845A实施例8)和VHH-IgG4-Fc纯化后,按照上文“(二)纳米抗体样品的外观及可见异物检测”所述程序进行观测,发现,纯化后的抗体样品均出现明显的絮状物。按照上文“(三)纳米抗体样品的CE-SDS检测”程序进行检测,发现均检测到分裂峰,结果分别见图1中的1A(VHH-IgG1-Fc)和1B(VHH-IgG4-Fc)。
不受限于任何理论,认为可能由于VHH、铰链区和Fc区域的特定氨基酸造成纯化后絮状物产生,且均一性很差,须进一步从成药性研发角度对现有序列进行优化。
实施例2已知抗PD-1纳米抗体的序列优化设计及初步筛选
为解决PR抗体纯化后出现絮状物及均一性较差的缺陷,对PR抗体进行优化,优化设计方案见表1。
表1.PR抗体的优化方案
优化的结构域 | 优化数(种) |
CDR | 2 |
FR | 8 |
FR+CDR | 12 |
FR+Hinge(铰链区) | 1 |
FR+polypeptide(连接肽) | 3 |
构型变化(Fc+VHH,VHH连至Fc的C端) | 1 |
注:表1中CDR,优化数2,是指针对CDR区设计了2种序列优化方案。同理,针对FR区设计了8种序列优化方案;同时针对FR和CDR区两者设计了8种序列优化方案;同时针对FR和Hinge(铰链区)区两者设计了1种序列优化方案;对FR序列优化的同时增加连接polypeptide(连接肽)的方案设计了3种;构型变化即VHH由原来的连接至Fc的N端改为连接至C端的方案。
按照上文“(一)纳米抗体样品的制备”所述程序制备优化纳米抗体样品,并按照上文“(二)纳米抗体样品的外观及可见异物检测”和“(三)纳米抗体样品的CE-SDS检测”程序进行样品检测。结果见表2。
表2.优化纳米抗体样品的检测结果
从CE-SDS检测结果及纯化后样品外观情况可以看出,N7、N10、N12、N13、N14、N15均一性好,且无明显絮状物形成。
实施例3优化纳米抗体的理化、生化分析
对初步筛选出的N7、N10、N12、N13、N14、N15进行理化、生化分析及进一步筛选。PR抗体与优化纳米抗体的序列见下(加粗、斜体并带下划线示出的为优化位点;按照IMGT定义CDR区域),序列比对见图2。PR:
VHH(SEQ ID NO.1)(CDR1/CDR2/CDR3:SEQ ID NO.2/SEQ ID NO.3/SEQ ID NO.4,以下划线并加粗示出):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以加粗示出):
N7:
VHH(SEQ ID NO.7)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.27/SEQ IDNO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR2突变氨基酸以下划线、斜体并加粗示出):
IgG4-Fc(SEQ ID NO.8)(铰链区:SEQ ID NO.9,以加粗示出,铰链区突变氨基酸以下划线示出):
VHH+铰链区(SEQ ID NO.10):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCP
抗体全长(SEQ ID NO.11):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N10:
VHH(SEQ ID NO.7)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.27/SEQ IDNO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR2突变氨基酸以下划线、斜体并加粗示出):
连接肽(SEQ ID NO.12):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以斜体并加粗示出):
VHH链+连接肽+铰链区(SEQ ID NO.13):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCP
抗体全长(SEQ ID NO.14):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N12:
VHH(SEQ ID NO.15)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.31/SEQ IDNO.2/SEQ ID NO.32/SEQ ID NO.3/SEQ ID NO.33/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR1和FR3突变氨基酸以下划线、斜体并加粗示出):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以加粗示出):
VHH链+铰链区(SEQ ID NO.16):
QVQLQESGGGLVQPGGSLRLSSAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYSAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSESKYGPPCPPCP
抗体全长(SEQ ID NO.17):
QVQLQESGGGLVQPGGSLRLSSAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYSAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N13:
VHH(SEQ ID NO.18)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.34/SEQ IDNO.2/SEQ ID NO.32/SEQ ID NO.3/SEQ ID NO.35/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR1和FR3突变氨基酸以下划线、斜体并加粗示出):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以加粗示出):
VHH链+铰链区(SEQ ID NO.19):
QVQLQESGGGLVQPGGSLRLSAAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYAAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSESKYGPPCPPCP
抗体全长(SEQ ID NO.20):
QVQLQESGGGLVQPGGSLRLSAAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYAAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N14:
VHH(SEQ ID NO.7)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.27/SEQ IDNO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR2突变氨基酸以下划线、斜体并加粗示出)
连接肽(SEQ ID NO.21):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以加粗示出):
VHH链+连接肽+铰链区(SEQ ID NO.22):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCP
抗体全长(SEQ ID NO.23):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N15:
VHH(SEQ ID NO.7)(FR1/CDR1/FR2/CDR2/FR3/CDR3/FR4:SEQ ID NO.27/SEQ IDNO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30,其中CDR1、CDR2、CDR3以下划线并加粗示出,FR2突变氨基酸以下划线、斜体并加粗示出)
连接肽(SEQ ID NO.24):
IgG4-Fc(SEQ ID NO.5)(铰链区:SEQ ID NO.6,以加粗示出):
VHH链+连接肽+铰链区(SEQ ID NO.25):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCP
抗体全长(SEQ ID NO.26):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
3.1 CE-SDS检测
按照上文“(二)纳米抗体样品的CE-SDS检测”程序进行样品检测。结果见表3和图3中的3A(PR)、3B(N7)、3C(N10)、3D(N12)、3E(N13)、3F(N14)和3G(N15)。
表3.优化纳米抗体样品的CE-SDS检测结果
3.2 SEC检测
按照上文“(四)纳米抗体样品的SEC检测”程序进行样品检测。结果见表4和图4中的4A(N7)和4B(N15)。
表4.优化纳米抗体样品的SEC检测结果
样品ID | 单体% |
PR | 90.7 |
N7 | 94.5 |
N10 | 96.4 |
N12 | 93.8 |
N13 | 96.2 |
N14 | 97.5 |
N15 | 97.9 |
3.3抗原结合(ELISA)
通过ELISA检测优化纳米抗体对抗原PD-1蛋白的结合能力。方法如下:
a.抗原包被:取100μL/孔浓度为20ng/mL的人PD-1蛋白(Acro PD1-HB2F2)于96孔板中,室温孵育2小时后,洗板5次;
b.参比品及供试品准备:参比品和供试品稀释至1000ng/mL后,进行梯度稀释;
c.反应:将梯度稀释后的样品按100μL/孔加入到步骤a中预先包被的96孔板中,盖上板盖,室温孵育2小时后,洗板5次;
d.加入检测抗体:按100μL/孔将稀释后的检测抗体转移至步骤c中样品板中,盖上盖板,室温孵育1小时后,洗板5次;
e.加入显色底物:在样品板中加入TMB显色底物,盖上盖板,室温孵育10-15分钟;
f.终止显色反应:在样品板中加入2M硫酸,终止显色反应;
g.读板:将样品板放置于酶标仪中,进行读板。
结果见表5。
表5.优化纳米抗体的抗原结合检测结果
样品ID | EC50(ng/ml) |
PR | 15.89 |
N7 | 9.98 |
N10 | 14.89 |
N12 | 15.30 |
N13 | 19.92 |
N14 | 15.12 |
N15 | 11.07 |
3.4抗原活性的阻断检测
检测优化纳米抗体对抗原PD-1蛋白活性的阻断作用。方法如下:
a.在1L的DMEM/F12基础培养基中加入FBS 100mL、NEAA 10mL、青链霉素溶液10mL、Hygromycin B(50mg/L)4mL和Puromycin(10mg/L)800μL,配制CHO-PD-L1-CD3L细胞完全培养基;将CHO-PD-L1-CD3L细胞接种到培养基中,每2天进行细胞传代培养至对数生长期;
在1640基础培养基1L中加入FBS 100mL、NEAA 10mL、青链霉素溶液10mL、Hygromycin B(50mg/L)8mL和Puromycin(10mg/L)400μL,配制Jurkat-PD-1-NFAT细胞完全培养基;将Jurkat-PD-1-NFAT细胞接种到培养基中,每2天进行细胞传代培养至对数生长期;
b.消化并离心CHO-PD-L1-CD3L细胞,用DMEM/F12完全培养基重悬细胞至5×105个/mL,每孔100μL加入到白底96孔板中,于37±2℃、(5±1)%CO2培养箱中孵育16±2h;
c.将供试品、参比品及质控品预稀释至100μg/mL,再用分析培养基(RPMI1640基础培养基+2%FBS)进行梯度稀释;
d.将步骤b的细胞培养板取出,吸取上清液弃掉,加入稀释后的供试品、参比品及质控品,每孔50μL;
e.取出Jurkat-PD-1-NFAT细胞,用分析培养基重悬细胞至2×106个/mL;以每孔50μL将该Jurkat细胞悬液加入到步骤d的培养板中,于培养箱中孵育6h;
f.提前1h取出Bio-Glo Luciferase Assay System试剂,室温放置,每孔100μL加入到细胞板;室温避光孵育2~3min后用酶标仪进行读数。
结果见表6。
表6.优化纳米抗体对抗原活性的阻断作用的检测结果
3.5结合亲和力(fortebio)
检测优化纳米抗体对抗原PD-1蛋白的结合亲和力(fortebio)。方法如下:
检测仪器:Octet K2,pall-fortebio
芯片:Protein A(厂家:Pall Fortebio,货号:18-5010)
缓冲液:pH 7.4PBST(pH 7.4 PBS,Tween20 0.05%v/v)
软件版本:fortebio data analysis 10.0
通过Protein A芯片特异性捕获待测抗体(PR,N7,N10,N12,N13,N14,N15),信号达到3nm,与梯度稀释的不同浓度PD-1蛋白进行结合。
其中,待测样品蛋白用PBST缓冲液稀释至终浓度为5ug/mL。将冻干PD-1蛋白用ddH2O稀释为250ug/mL,然后用PBST稀释,最高浓度为50nM,2倍浓度梯度稀释,共7个浓度。
结果见表7。
表7.优化纳米抗体对抗原的结合亲和力
将3.1至3.5项检测数据结果汇总于表8。
表8.优化纳米抗体的理化、生化分析结果
实施例4优化纳米抗体的抑瘤作用检测
在C57BL/6hPD1人源化小鼠中检测本发明的优化纳米抗体对皮下肿瘤的治疗作用。
在C57BL/6J-hPD1人源化小鼠皮下移植MC38-hPD-L1肿瘤细胞(结肠癌细胞),建立MC38-hPD-L1移植瘤模型。方法如下:
1.试验处理前小鼠进行隔离与适应性饲养一周;
2.将MC38-hPD-L1细胞在37℃、5%CO2的培养箱中培养扩增,收取对数生长期细胞重悬于PBS中,加入基质胶,调整细胞浓度到1×107个细胞/mL;
3.用1mL注射器将细胞悬液注入C57BL/6J-hPD1人源化小鼠右侧皮下,每只小鼠注射100μL;
4.待平均肿瘤体积约为112mm3时,淘汰体积过大、过小或者肿瘤形状不规则的小鼠。
采用随机区组法将小鼠分为5组,分别为对照组(组1)、Keytruda(组2)、PR抗体组(组3)、优化纳米抗体N7组(组4)、优化纳米抗体N15组(组5),每组8只。按照表9中的方案给药,各组均采用腹腔给药,各组给药体积均为10ml/kg,连续给药3周,每周给药2次,共给药6次。其中各组抗体配制成1mg/ml的使用浓度,对照组采用生理盐水,分组当天开始给药。
表9.给药方案
每周测量2次瘤径、称量小鼠体重,观察小鼠生活状态,对异常情况进行记录。采用以下计算公式:
1.肿瘤体积(Tumor volume,TV)
TV=1/2×a×b2
其中:a表示肿瘤长径;b表示肿瘤短径。
2.相对肿瘤体积(Relative tumor volume,RTV)
RTV=Vt/Vinitial×100(%)
其中,Vinitial为分组给药时(即dinitial)测量所得肿瘤体积,Vt为每次测量时的肿瘤体积。
3.相对肿瘤增殖率T/C(%)
T/C(%)=(TRTV/CRTV)×100%
其中,TRTV表示治疗组的相对肿瘤体积,CRTV表示溶剂组的相对肿瘤体积。
4.肿瘤体积抑瘤率(TGI)
TGI=[1-(TVt-TVinitial)/(CVt-CVinitial)]×100%
其中,TVt表示治疗组每次测量时的肿瘤体积;TVinitial表示分组给药时治疗组的肿瘤体积;CVt表示对照组每次测量时的肿瘤体积;CVinitial表示分组给药时对照组的肿瘤体积。
结果用平均数和标准误(Mean±SEM)表示,使用T-test分析对照组和给药组的肿瘤体积差异,p<0.05表示存在统计学差异。
一、小鼠肿瘤体积变化见表10和图5。
表10.小鼠肿瘤体积(mm3)
注:数据用Mean±SEM表示
“-”:动物安乐死
给药开始后第14天,对照组平均肿瘤体积为4057.43±288.73mm3。组2、组3、组4和组5肿瘤体积分别为884.86±194.57mm3、1584.55±186.82mm3、672.45±101.01mm3和815.51±131.88mm3,肿瘤抑制率分别为80.42%、62.69%、85.79%和82.16%。组2、组3、组4和组5与对照组肿瘤体积比较,有显著性差异(P<0.01)。
以第14天测得的肿瘤体积为基础,计算抗体对移植hPD-L1肿瘤的雌性人源化小鼠的肿瘤生长抑制能力。结果见表11和表12。
表11.肿瘤生长抑瘤率分析
表12.各组之间肿瘤体积比较
注:数据用Mean±SEM表示
采用T-test分析比较vehicle组和治疗组之间的肿瘤体积,****表示p<0.0001,*表示p<0.05。
二、小鼠肿瘤重
试验结束时,对动物实施安乐死,剥取瘤块称重,小鼠瘤重情况见图6,具体的,对照组平均肿瘤重为7.0907±0.8349g,受试样品Keytruda、PR、N7和N15平均肿瘤重分别为3.0556±0.9384g、5.1787±0.3078g、2.4394±0.7359g和3.0210±0.8778g。
表10至表12及图5和图6的实验结果表明,除受试物PR外,受试物N7、N15和Keytruda抗体单独治疗在hPD-L1-MC38肿瘤模型上均相对对照组取得了优异的抗肿瘤效果。N7、N15、Keytruda在抗肿瘤效果上显著优于PR抗体,N7、N15相较Keytruda在hPD-L1-MC38肿瘤模型上显示相当的抗肿瘤效果。鉴于Keytruda作为PD-1/PD-L1单抗类药物已被国内外药监机构批准用于多达13种肿瘤或癌症的治疗,给患者带来了巨大的临床获益,也取得了巨大商业成功,本发明抗PD-1纳米抗体作为药效不逊于其的可替代的纳米抗体药物也具有广阔的临床治疗前景。另外本发明抗PD-1纳米抗体提供了相较PR而言,更符合药物开发要求的药物候选分子。
以上对本发明具体实施方式的描述并不限制本发明,本领域技术人员可以根据本发明作出各种改变或变形,只要不脱离本发明的精神,均应属于本发明所附权利要求的范围。
序列表
<110> 浙江特瑞思药业股份有限公司
<120> 一种抗PD-1纳米抗体及其应用
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<223> N7
<400> 11
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 Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Arg Lys Ser Ser Val Glu Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
225 230 235 240
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Leu Gly Lys
355
<210> 12
<211> 28
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> linker
<400> 12
Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro Gln
1 5 10 15
Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu
20 25
<210> 13
<211> 169
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH+linker+hinge
<400> 13
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 Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro
130 135 140
Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu Glu Ser Lys
145 150 155 160
Tyr Gly Pro Pro Cys Pro Pro Cys Pro
165
<210> 14
<211> 386
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> N10
<400> 14
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 Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro
130 135 140
Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu Glu Ser Lys
145 150 155 160
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
165 170 175
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
180 185 190
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
195 200 205
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
210 215 220
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
225 230 235 240
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
245 250 255
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
260 265 270
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
275 280 285
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
290 295 300
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
305 310 315 320
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
325 330 335
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
340 345 350
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
355 360 365
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
370 375 380
Gly Lys
385
<210> 15
<211> 129
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH
<400> 15
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 Ser Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 16
<211> 141
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH+hinge
<400> 16
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 Ser Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
130 135 140
<210> 17
<211> 358
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> N12
<400> 17
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 Ser Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
225 230 235 240
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Leu Gly Lys
355
<210> 18
<211> 129
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH
<400> 18
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 Ala Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ala Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 19
<211> 141
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH+hinge
<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 Ala Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ala Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
130 135 140
<210> 20
<211> 357
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> N13
<400> 20
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 Ala Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ala Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
130 135 140
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
225 230 235 240
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Leu Gly Lys
355
<210> 21
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> linker
<400> 21
Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys
1 5 10
<210> 22
<211> 151
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH+linker+hinge
<400> 22
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 Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Glu Ser Lys Tyr Gly
130 135 140
Pro Pro Cys Pro Pro Cys Pro
145 150
<210> 23
<211> 368
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> N14
<400> 23
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 Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Glu Ser Lys Tyr Gly
130 135 140
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
145 150 155 160
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
165 170 175
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
180 185 190
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
195 200 205
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
210 215 220
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
225 230 235 240
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
245 250 255
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
260 265 270
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
275 280 285
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
290 295 300
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
305 310 315 320
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
325 330 335
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
340 345 350
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
355 360 365
<210> 24
<211> 18
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> linker
<400> 24
Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu
1 5 10 15
Pro Glu
<210> 25
<211> 159
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH+linker+hinge
<400> 25
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 Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro
130 135 140
Glu Pro Glu Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
145 150 155
<210> 26
<211> 376
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> N15
<400> 26
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 Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro
130 135 140
Glu Pro Glu Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
145 150 155 160
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
165 170 175
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
180 185 190
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
195 200 205
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
210 215 220
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
225 230 235 240
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
245 250 255
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
260 265 270
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
275 280 285
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
290 295 300
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
305 310 315 320
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
325 330 335
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
340 345 350
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
355 360 365
Ser Leu Ser Leu Ser Leu Gly Lys
370 375
<210> 27
<211> 25
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH, FR1
<400> 27
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
20 25
<210> 28
<211> 15
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH, FR2
<400> 28
Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Ala
1 5 10 15
<210> 29
<211> 38
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH, FR3
<400> 29
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Asp Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 30
<211> 11
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH, FR4
<400> 30
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 31
<211> 25
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH, FR1
<400> 31
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 Ser Ala Ala Ser
20 25
<210> 32
<211> 15
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH, FR2
<400> 32
Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val Ala Ala
1 5 10 15
<210> 33
<211> 38
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH, FR3
<400> 33
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Asp Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Ser
35
<210> 34
<211> 25
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH, FR1
<400> 34
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 Ala Ala Ala Ser
20 25
<210> 35
<211> 38
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> VHH, FR3
<400> 35
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Asp Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Ala
35
Claims (8)
1.一种抗PD-1纳米抗体,其特征在于,所述抗PD-1纳米抗体由VHH链与包含经修饰铰链区的IgG4-Fc组成,其中:
(i) 所述抗PD-1纳米抗体VHH链包含SEQ ID NO. 2所示的CDR1、SEQ ID NO. 3所示的CDR2、SEQ ID NO. 4所示的CDR3及SEQ ID NO. 28所示的CDR1和CDR2之间的框架区FR2;
并且
(ii) 所述抗PD-1纳米抗体中的经修饰铰链区由连接肽与SEQ ID NO: 6所示的IgG4-Fc铰链区序列组成,所述连接肽的氨基酸序列如SEQ ID NO. 12、SEQ ID NO. 21或SEQ IDNO. 24所示并且连接至SEQ ID NO. 6所示的IgG4-Fc铰链区的N端。
2.根据权利要求1所述的抗PD-1纳米抗体,其特征在于,所述抗PD-1纳米抗体为抗人PD-1纳米抗体。
3.权利要求1或2所述的抗PD-1纳米抗体在制备用于治疗肿瘤的药物中的用途,其特征在于,所述肿瘤为肺癌、胃癌、肝癌、黑色素瘤、宫颈癌、结直肠癌、膀胱癌、乳腺癌、淋巴瘤、肾细胞癌、盲肠癌、胰腺癌、胆管癌、头颈癌、梅克尔细胞癌、卵巢癌、鼻咽癌、胶质瘤、食管癌、骨癌或前列腺癌。
4.根据权利要求3所述的用途,其特征在于,所述肿瘤为结直肠癌。
5.权利要求1或2所述的抗PD-1纳米抗体在制备PD-1检测试剂中的用途。
6.一种PD-1检测试剂,其特征在于,所述PD-1检测试剂包含权利要求1或2所述的抗PD-1纳米抗体。
7.根据权利要求6所述的PD-1检测试剂,其特征在于,所述PD-1检测试剂为基于流式细胞术或酶联免疫吸附法的检测试剂。
8.一种试剂盒,其特征在于,所述试剂盒包含权利要求6或7所述的PD-1检测试剂。
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