CN112409485A - 抗CEACAM6单域抗体、人源化单域抗体及其Fc融合蛋白和应用 - Google Patents
抗CEACAM6单域抗体、人源化单域抗体及其Fc融合蛋白和应用 Download PDFInfo
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- CN112409485A CN112409485A CN202011135295.5A CN202011135295A CN112409485A CN 112409485 A CN112409485 A CN 112409485A CN 202011135295 A CN202011135295 A CN 202011135295A CN 112409485 A CN112409485 A CN 112409485A
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Abstract
本发明公开了抗CEACAM6单域抗体、人源化单域抗体及其Fc融合蛋白和应用。本发明筛选得到一组具有较强的中和或结合能力的抗CEACAM6单域抗体，能特异性结合CEACAM6。本发明还将所述单域抗体进行人源化改造得到亲和力提高的人源化抗体。本发明进一步将所述单域抗体或人源化后的单域抗体与人IgG‑Fc融合得到单域抗体‑Fc融合蛋白，融合蛋白用同位素标记后能很好的特异性靶向小鼠体内移植肿瘤。本发明提供的抗CEACAM6单域抗体、人源化单域抗体、单域抗体‑Fc融合蛋白可用于检测或诊断CEACAM6以及用于治疗CEACAM6表达异常相关疾病。
Description
技术领域
本发明涉及单域抗体，尤其涉及抗CEACAM6的单域抗体以及用该单域抗体或人源化的单域抗体与IgG1-Fc融合构建的融合蛋白，本发明进一步涉及它们在检测CEACAM6以及治疗CEACAM6表达异常相关疾病中的应用，属于抗CEACAM6的单域抗体、人源化的单域抗体及其应用领域。
背景技术
多种癌胚抗原相关细胞粘附分子(Carcinoembryonic antigen(CEA)relatedcell adhesion molecule(CEACAM))的蛋白属于免疫球蛋白(Ig)超基因的家族成员，其蛋白组成的一级结构为细胞的胞外区、跨膜区和胞内区(有些成员没有胞内区)。常见家族成员有CEACAM1,3,4,5,6,7,8,16,18,19,21，其蛋白的胞外结构域的特点是：有N端的N结构域、随后为无或1-6个恒定C2样Ig结构域(称为A区或B区)。
癌胚抗原相关细胞粘附分子的这些胞外结构域作为同嗜性和异嗜性细胞间粘附分子或作为人类和啮齿类病原体受体是CEACAM展示其功能所必需的。CEACAM受体可以是寡聚体或二聚体与其它配体在细胞膜形成多重组合从而调节其细胞重要功能。除了在人类组织中的表达外，CEACAM基因家族在27种其他哺乳动物物种中高度保守(Robert Kammerer,Wolfgang Zimmermann.Coevolution of activating and inhibitory receptors withinmammalian carcinoembryonic antigen families.BMC Biol.2010Feb 4；8:12)。CEACAM的生物功能是通过其同嗜性和异嗜性相互作用来维系细胞与细胞粘附，包括在三维组织结构的分化和形成、血管生成、细胞凋亡、肿瘤抑制和转移中的作用等(Kuespert K.etal.CEACAMs:their role in physiology and pathophysiology.Curr Opin CellBiol.2006Oct；18(5):565-71；Athanasia Pavlopoulou and Andreas Scorilas.AComprehensive Phylogenetic and Structural Analysis of the CarcinoembryonicAntigen(CEA)Gene Family.Genome Biol Evol.2014Jun；6(6):1314–1326)。
癌胚抗原相关细胞粘附分子6(CEACAM6)，又称为非特异性交叉反应抗原(non-specific cross-reacting antigen(NCA，NCA-50/90))、CD66c是癌胚抗原相关细胞粘附分子蛋白家族的重要成员之一，其与家族成员CEACAM1/7/8的分子有高度同源性。CEACAM6是具有一个N结构域和2个C2样结构域的糖基磷脂酰肌醇(glycosylphosphoinositol,GPI)-连接的细胞表面蛋白，通过其具有各种膜受体(已鉴定其中的一些)的胞外结构域介导许多可能的顺式或反式引导的CEACAM相互作用。已有研究报道CEACAM6在多种肿瘤中过表达，如在非小细胞肺癌、胰腺癌、乳腺癌、结直肠癌、肝癌、胃癌、还有卵巢癌等都有不同程度的过表达。CEACAM6的过表达，能导致上皮组织中叶间质化样形态学改变、肿瘤的侵袭性和对抗化学药物的耐受性增强、肿瘤的转移、以及减少细胞凋亡等，用SiRNA减少CEACAM6的基因表达和用单克隆抗体抑制CEACAM6蛋白的功能，能使CEACAM6过表达所产生的这些作用逆转。虽然CEACAM6在人的许多正常组织中如粒细胞系等也有所表达，但在多种肿瘤中过表达，已有许多研究报道；有比较肺癌、乳腺癌、前列腺癌、结肠癌、胰腺癌和卵巢癌组织及其肿瘤组织旁组织和正常组织的CEACAM6与CEACAM5(CEA)表达程度的研究显示：在所有研究的肿瘤类型中，CEACAM6都比CEA表达高，而且在CEACAM6过表达的癌症中，不同组织类型的肿瘤，CEACAM6的表达丰度也有所不同，如在乳腺肿瘤CEACAM6的表达丰度为：乳突状癌＞侵润导管型＞小叶状＞叶状柄型；在胰腺癌CEACAM6的表达丰度为：中度分化型＞充分分化型＞低分化型肿瘤；黏液型卵巢腺癌的CEACAM6的表达比浆液型卵巢腺癌高3倍；在非小细胞肺癌CEACAM6的表达是肺腺癌＞肺鳞癌；在肝转移结肠癌的CEACAM6表达＞原发肿瘤＞淋巴结转移肿瘤。前列腺癌组织的CEACAM6的表达与其癌旁正常组织的表达没有区别(NicodeBeauchemin andAzadehArabzadeh.Carcinoembryonic antigen-related cell adhesionmolecules(CEACAMs)in cancer progression and metastasis.Cancer MetastasisRev.2013 Dec.；32(3-4):643-71；Rosalyn D Blumenthal et al.Expression patternsof CEACAM5 and CEACAM6 in primary and metastatic cancers.BMC Cancer.2007 7:2(1-15))。
如上所述，CEACEA6可能是这些过表达肿瘤的一个特异性靶抗原，CEACAM6是癌症免疫治疗中的治疗干预的非常有吸引力的一个新靶标。
单域抗体(single domain antibody(sdAb)或称纳米抗体(nanobody)是由羊驼血液中发现的缺失轻链的重链抗体可变区片段(VHH)，其具有结构简单，穿透力强，易于表达和纯化，亲和力及稳定性高，毒副反应小等一系列优点。利用单域抗体技术筛选得到亲和力高的抗CEACAM6单域抗体，能用于CEACAM6过表达肿瘤的检测和治疗研究，还可为CEACAM6过表达肿瘤的相关疾病提供新检测方法和治疗手段。
发明内容
本发明的目的之一是提供一组抗CEACAM6的单域抗体及其编码基因；
本发明的目的之二是将抗CEACAM6的单域抗体进行人源化改造得到人源化的单域抗体；
本发明的目的之三是将所述的单域抗体或人源化的单域抗体与人IgG1-Fc融合得到融合蛋白；
本发明的目的之四是将所述的单域抗体或人源化的单域抗体与酶相、放射性同位素、荧光化合物或化学发光化合物中的一种或多种相偶联得到缀合物；
本发明的目的之四将所述的抗CEACAM6的单域抗体、抗CEACAM6的人源化的单域抗体、融合蛋白以及缀合物应用于制备检测CEACAM6的试剂或治疗CEACAM6表达异常相关疾病；
本发明的上述目的是通过以下技术方案来实现的：
本发明首先提供了一组抗CEACAM6的单域抗体，所述单域抗体均由框架区和3个互补决定区组成，所述单域抗体选自NBC10、NBC25或NBC36中的任何一种；其中，单域抗体NBC10的3个互补决定区的氨基酸序列分别为SEQ ID No.1、SEQ ID No.2和SEQ ID No.3所示；单域抗体NBC25的3个互补决定区的氨基酸序列分别为SEQ ID No.4、SEQ ID No.5和SEQID No.6所示；单域抗体NBC36的3个互补决定区的氨基酸序列分别为SEQ ID No.7、SEQ IDNo.8和SEQ ID No.9所示；
本发明进一步提供了所述单域抗体的氨基酸序列，其中，单域抗体NBC10的氨基酸序列为SEQ ID No.10所示，单域抗体NBC25的氨基酸序列为SEQ ID No.11所示，单域抗体NBC36的氨基酸序列为SEQ ID No.12所示。
将上述所示的任何一种氨基酸序列中的一个或多个氨基酸进行缺失、取代、插入和/或添加所得到的蛋白突变体，该蛋白突变体与突变前的蛋白具有相同的功能，这些蛋白突变体均属本发明的保护范畴之内；此外，与上述所示的任何一种氨基酸序列至少有90％以上同一性的氨基酸序列也属于本发明保护范畴之内。
本发明还进一步提供了所述单域抗体的编码基因序列，其中，单域抗体NBC10的编码基因的核苷酸序列为SEQ ID No.13所示，单域抗体NBC25的编码基因的核苷酸序列为SEQID No.14所示，单域抗体NBC36的编码基因的核苷酸序列为SEQ ID No.15所示。其中，与上述所示的多核苷酸序列的互补序列在严谨杂交条件能够进行杂交的多核苷酸序列也属于本发明的保护范畴之列；另外，与上述任何一种所示的多核苷酸序列至少有90％以上同一性的多核苷酸序列也属于本发明的保护范畴之列。
本发明进一步提供了一种重组表达载体，所述重组表达载体包含所述单域抗体的编码基因中的一个或多个；优选的，所述重组表达载体可以是重组原核细胞表达载体、重组酵母表达载体、重组真核细胞表达载体或其它重组细胞表达载体。
本发明还提供了重组宿主细胞，其包含上面所述的重组表达载体。
优选的，所述的重组宿主细胞为重组原核表达细胞、重组真核表达细胞，重组真菌细胞或酵重组母细胞，所述重组原核表达细胞优选大肠杆菌。
本发明进一步将单域抗体NBC10进行了人源化改造得到了5个人源化的抗体NBC10HM1、NBC10HM2、NBC10HM3、NBC10HM4和NBC10HM5，其氨基酸序列分别为SEQ ID No.16、SEQ ID No.17、SEQ ID No.18、SEQ ID No.19和SEQ ID No.20所示。
本发明进一步将单域抗体NBC36进行了人源化改造得到了2个人源化的抗体NBC36HM1和NBC36HM2，其氨基酸序列分别为SEQ ID No.21和SEQ ID No.22所示。
本发明进一步将所述的抗CEACAM6的单域抗体或人源化的单域抗体与IgG-Fc构建融合蛋白；其中，所述Fc基因序列可以是来源于IgG，IgA，IgM的Fc基因序列或来源于IgG1，IgG2，IgG3或IgG4。所述的IgG优选是人IgG及其IgG1、2、3、4的亚类，还可以是人IgM、人IgA或其它动物(如鼠，兔，猴等)免疫球蛋白的Fc片段基因及氨基酸序列。
作为本发明一种优选的实施方式，本发明将人源化的抗体NBC25HM3与人IgG1-Fc基因进行融合得到氨基酸序列为SEQ ID No.23所示的融合蛋白，其编码基因的核苷酸序列为SEQ ID No.24所示；或者将将人源化的抗体NBC36HM2与人IgG1-Fc基因进行融合得到氨基酸序列为SEQ ID No.25所示的融合蛋白，其编码基因的核苷酸序列为SEQ ID No.26所示。
本发明进一步将所述单域抗体或人源化的单域抗体与酶相(如辣根过氧化物酶、碱性磷酸酶等)、放射性同位素、荧光化合物或化学发光化合物(所述的化学发光化合物可以是荧光化合物)中的一种或多种相偶联得到缀合物，这些缀合物可用于检测CEACAM6或治疗多种与CEACAM6表达异常相关疾病。
譬如，将抗CEACAM6人源化的单域抗体、Fc融合蛋白用68Ga,89Zr,64Cu,18F,86Y,90Y,111In,99NVTc,125I,124I等放射性同位素进行标记得到标记蛋白用于PET(positronemission tomography)或SPECT的影像学检测。或者将抗CEACAM6人源化的单域抗体、Fc融合蛋白用90Y，177Lu,125I,131I,211At,111In,152Sm,186Re,188Re,67Cu,212Pb,225Ac,213Bi,212Bi或67Ga等放射性同位素进行标记得到标记蛋白用于CEACAM6表达异常相关疾病的治疗。
本发明所提供的抗CEACAM6的单域抗体、人源化的抗CEACAM6的单域抗体、或人源化的单域抗体与IgG-Fc构建的融合蛋白、单域抗体或人源化的单域抗体与酶相、放射性同位素、荧光化合物或化学发光化合物相偶联得到缀合物主要具有如下几方面的用途：
(1)制备检测CEACAM6有关的药物或试剂；
(2)制备用于治疗CEACAM6表达异常相关疾病的药物的应用；优选的，所述的CEACAM6表达异常相关疾病包括非小细胞肺癌、胰腺癌、乳腺癌和卵巢癌等肿瘤疾病。
本发明所涉及的术语定义
本文所用的术语“CEACAM6”，癌胚抗原相关细胞粘附分子6(CEACAM6)，又称为非特异性交叉反应抗原(non-specific cross-reacting antigen(NCA，NCA-50/90))、CD66c是癌胚抗原相关细胞粘附分子蛋白家族的重要成员之一。CEACAM6是具有一个N结构域和2个C2样结构域的糖基磷脂酰肌醇(glycosylphosphoinositol,GPI)-连接的细胞表面蛋白，通过其具有各种膜受体(已鉴定其中的一些)的胞外结构域介导许多可能的顺式或反式引导的CEACAM相互作用。已有研究报道CEACAM6在多种肿瘤中过表达，如在非小细胞肺癌、胰腺癌、乳腺癌、结直肠癌、肝癌、胃癌、还有卵巢癌等都有不同程度的过表达。CEACEA6可能是这些过表达肿瘤的一个特异性靶抗原，CEACAM6是癌症免疫治疗中的治疗干预的非常有吸引力的靶标。
针对CEACAM6的新抗体及其Fc融合蛋白是本文的研发对象，最终也是本文的保护对象，本文的范围涉及所得到的抗CEACAM6人源化单域抗体及其Fc融合蛋白以该抗体为成分的物质(如，药物组合物、试剂盒、载体等等)、应用(如，诊断应用、治疗应用、制备应用等等)，然而，本领域技术人员应该理解，本文的保护对象并不限于举例的这些内容。
本文所用的术语“单域抗体(sdAb)”是指包含了抗体中单个可变域的片段，也称为纳体(Nanobody)。和完整的抗体一样，它可以选择性的和特定抗原结合。与完整抗体的150－160kDa的质量相比，单域抗体则显得小得多，大约只有12－17kDa。第一个单域抗体是从骆驼的重链抗体中人造工程制作出来的，称为“VHH区段”。
本文所用的术语序列的“同一性(identity)”可以与“相同性”互换使用，指的是序列之间通过序列比对软件例如BLAST确定的相似程度。序列比对的方法和软件对于本领域技术人员是公知的。可以通过对已知序列进行一个或几个氨基酸或碱基的取代、缺失和/或添加而获得经改造的核苷酸序列。例如，通过常规手段(例如保守取代等)，对本发明的序列SEQ ID NO：1-198中一个或多个所示的氨基酸或核苷酸序列进行改造，可以获得与这些具有大于80％、大于85％、大于90％、大于95％或大于99％的序列同一性，并且具有基本相同的性能，这都在本发明的保护范围之内。优选地，本发明通过保守取代获得序列同一性，但并不限于保守取代。
术语“互补的”在此指的是两种包括反向平行核苷酸序列的核苷酸序列，反向平行核苷酸序列能在反向平行核苷酸序列的互补碱基残基之间形成氢键后彼此相互配对。本领域已知的是，当都从5’到3’的方向看序列时，两种互补链的核苷酸序列是彼此反向互补的。本领域也已知的是，两种在给定的条件组下能彼此杂交的序列不必必须是100％完全互补的。
术语“氨基酸序列”是指氨基酸相互连接形成肽链(或多肽)的顺序，氨基酸序列只能按照一个方向读取。氨基酸有100多种不同类型，其中20种常用，本发明不排除氨基酸链上有其他物质，例如糖类、脂类等修饰，本发明也不限于20中常用的氨基酸。
术语“核苷酸序列”是指DNA或RNA中碱基的排列顺序，即在DNA中为A、T、G、C的排列顺序，或者在mRNA中A、U、G、C的排列顺序，也包括rRNA、tRNA、mRNA中碱基的排列顺序。应该理解，本发明请求保护的抗体基因除了DNA序列外，也涵盖RNA(rRNA、tRNA、mRNA)以及它们的互补序列。
本发明中所述的取代可以是保守取代，即将特定的氨基酸残基替换为具有相似物理化学特征的残基。保守取代的非限定性例子包括含脂肪族基团氨基酸残基之间的取代(例如Ile、Val、Leu或Ala间的相互取代)、极性残基之间的取代(例如Lys和Arg、Glu和Asp、Gln和Asn之间的相互取代)等。因氨基酸的缺失、取代、插入和/或添加而成的突变体可以通过对编码野生型蛋白质的DNA实施例如作为公知技术的定点诱变(参见例如Nucleic AcidResearch，Vol.10，No.20，p.6487-6500，1982，通过引用其全文引入本说明书)来制作。
术语“表达载体(Expression vectors)”是指在克隆载体基本骨架的基础上增加表达元件(如启动子、RBS、终止子等)，使目的基因能够表达的载体。表达载体四部分：目的基因、启动子、终止子、标记基因。本发明包括但不限于原核细胞表达载体、真核细胞表达载体或其它细胞表达载体。
术语“框架区(Framework region)”，即骨架区，在免疫球蛋白的H和L链的近N端约有110个氨基酸序列的变化很大，其他部分的氨基酸序列相对恒定，据此可将轻链和重链区分为可变区(V)和恒定区(C)。可变区内包含超变区HVR(hypervariable region)或称互补决定区CDR(Complementarity-determining region)与FR骨架区。
术语“人源化”抗体是指抗体的可变区(VH或VHH)的Fr区部分，恒定区部分(即CH和CL区)或抗体所有全部由人类抗体基因所编码。人源化抗体可以大大减少异源抗体对人类机体造成的免疫副反应。人源化抗体包括嵌合抗体、改型抗体和全人源化抗体等几类。应该理解，本领域技术人员根据实际需要能够制备出本发明单域抗体的合适的人源化形式，这在本发明涵盖的范围之内。
术语“突变”和“突变体”在此具有它们的常用含义，指的是在核酸或多肽序列中的遗传的、天然存在的或引入的变化，它们的意义与本领域人员通常所知的意义相同。
术语“宿主细胞”或“重组宿主细胞”意指包含本发明多核苷酸的细胞，而不管使用何种方法进行插入以产生重组宿主细胞，例如直接摄取、转导、f配对或所属领域中已知的其它方法。外源性多核苷酸可保持为例如质粒的非整合载体或者可整合入宿主基因组中。
附图说明
图1构建CEACAM6基因库，巢式PCR扩增第一轮PCR产物，在800～500bp之间的为缺失轻链的重链抗体基因片段。
图2用VHH特异性引物经PCR扩增得到VHH目的基因。
图3为表达的部分抗CEACAM6抗单域抗体蛋白SDS-PAGE电泳结果。
图4为表达的部分CEACAM6-sdAB经镍柱纯化后SDS-PAGE电泳结果。
图5为纯化的抗CEACAM6单域抗体与人CEACAM6抗原特异性结合的活性试验结果。
图6为3种CEACAM6人源化单域抗体表达纯化后SDS-PAGE还原胶和非还原胶电泳结果；1.EG2M1-EG10M1-Fc-p327.7表达纯化后还原性蛋白带；2.EG2M1-Fc-EG10M1-p327.7表达纯化后还原性蛋白带；3.EG2M1-EG10M1-Fc-p327.7表达纯化后非还原性蛋白带；4.EG2M1-Fc-EG10M1-p327.7表达纯化后非还原性蛋白带；5.蛋白质分子量标准(Marker)；箭头所示分子量为50KD。
图7抗体修饰及89Zr标记路线图。
图8单域抗体-Fc融合蛋白标记同位素89Zr在小鼠肿瘤动物模型体内重要器官和肿瘤部位组织的分布结果(PET/CT扫描图)。
图9给予89Zr-CEACAM637.2抗体后各时间点各组织放射性物质摄取％ID/g值柱状图。
具体实施方式
以下结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1抗CEACAM6抗原特异性的单域抗体库构建
(1)CEACAM6抗原免疫羊驼：按照常规免疫方法进行，以购买的CEACAM6抗原(HumanCEACAM6 Protein,Human,Recombinant(His Tag))，选择成年健康的羊驼，在颈背部皮下多点注射抗原，加入抗原和等体积福氏佐剂，分4-8次免疫，跟踪观察注射部位包块吸收情况，以确认免疫正确。第一次免疫后，间隔21天，从第二针免疫开始，免疫间隔时间为7-15天，第4次免疫后，采血清，测定抗原免疫效价，当效价达到5万倍左右时(ELISA方法)，采全血100ml左右，分离淋巴细胞，-80℃保存备用。
(2)羊驼外周血淋巴细胞的分离和RNA的提取：分离羊驼外周血白细胞，应用QIAGEN试剂盒按照说明书进行提取RNA。RNA纯化：应用QIAGEN试剂盒进行RNA纯化，按照说明书进行，测定得到的RNA浓度和OD260/280≥1.8。
(3)重链抗体可变区-VHH：合成cDNA第一链：用cDNA合成试剂盒(MiniBESTAgaroseGel DNA Extraction Kit Ver.4.0，TAKARA公司)，按照说明书进行。以此模板，分别用两套引物进行PCR扩增重链抗体VHH基因片段。采用巢式PCR方法，第一次PCR扩增中大于800bp的为普通重链基因片段，在800～500bp之间的为缺失轻链的重链抗体基因片段(图1)，切胶回收缺失轻链重链抗体基因片段，以此为模板用VHH特异性引物经PCR扩增得到VHH目的基因(～500bp)，基因扩增结果见图2。所用引物：
第一轮PCRFd5’引物:YF:CGC CAT CAA GGT ACC AGT TGA；
第一轮PCR Bd3’引物:YBN:CAG CCG GCC ATG GCC SMK GTR CAG CTG GTG GAKTCT GGG GGA G；
第二轮PCR引物：
YV-BACK：CAT GTG CATGGCCTA GAC TCG CGG CCCAGC CGG CCA TGG CC；YV-FOR：CAT GTG TAG ATT CCT GGC CGG CCT GGC CTG AGG AGA CGG TGA CCT GG；
(4)VHH片段和噬菌体展示载体的连接及电转化TG1感受态：SfI单酶切VHH片段和pHEN6载体质粒后，将VHH片段及pHEN6载体(Conrath,KEM other.Antimicrob AgentsChemother(Antimicrobial Chemotherapy)2001,45:(10)2807-12.)经连接酶(T4，NEB公司)连接，电转化至TG1感受态细胞中，做10个电转，涂布平板，经菌落PCR验证抗体插入率。重组基因克隆效率检测：取电转化菌液涂布LB/Amp平板上，32℃，过夜培养，次日用菌落PCR的方法验证抗体的连接效率，噬菌体抗体库的连接效率在90％以上。将电转化菌液涂布LB/Amp平板上，32℃，过夜培养物，用2YT培养基洗下，加入15％甘油，-80℃保存。噬菌体库1.8×108；随机挑选30-50个克隆，克隆PCR，VHH基因插入率95％，并进行基因测序，其VHH的序列中三个CDR序列重复率小于2％。
(5)VHH噬菌体抗体库的制备：抗体库加入辅助噬菌体M13K07(Invitrogen)进行拯救：按照常规方法进行噬菌体抗体库的制备，并保存于-80℃备用。
实施例2抗CEACAM6的单域抗体的筛选
(1)针对CEACAM6特异性单域抗体的筛选
第一轮CEACAM6蛋白浓度50μg/ml，0.5ml包被免疫管(Thermofisher公司)，4℃过夜。第二轮、第三轮分别用CEACAM6蛋白浓度20μg/ml、10ug/ml，0.5ml包被免疫管，4℃过夜。封闭：2％脱脂奶粉PBS，37℃，孵育1.5小时。加入噬菌体，室温1小时，用PBST和PBS各洗10次，用0.5ml的TEA，洗脱特异性结合的噬菌体，感染2ml对数生长期的TG1，测定滴度，并培养扩增噬菌体，进行新一轮筛选。
表1针对CEACAM6特异性单域抗体的筛选结果
| 筛选次数 | 加入噬菌体量 | 洗脱回收噬菌体量 |
| 第一轮 | 1.1×10<sup>12</sup> | 3.5×10<sup>5</sup> |
| 第二轮 | 1.2×10<sup>12</sup> | 4.3×10<sup>6</sup> |
| 第三轮 | 5.0×10<sup>11</sup> | 6.8×10<sup>7</sup> |
(2)噬菌体ELISA方法挑选阳性克隆
从第2和/或第3轮筛选生长在琼脂平板上的菌落，随机挑取单个菌落，接种在含有Amp的2YT液体培养基的96深孔培养板中培养，用辅助噬菌体超感染诱导表达噬菌体抗体。收获表达上清，以CEACAM6为抗原进行ELISA测定，挑选出CEACAM6阳性孔，经DNA测序以鉴定抗单域抗体克隆的基因序列，得到包括SEQ ID NO.13-15所示基因序列在内的一系列单域抗体基因序列，用于进一步表达和筛选特异性、高活性的单域抗体。
实施例3特异性CEACAM6单域抗体表达质粒的构建
PCR扩增实施例2所获得的特异性CEACAM6的单域抗体基因，获得带有限制性内切酶BbsI和BamHI位点PCR产物，用限制性内切酶BbsI和BamHI分别处理PCR产物和载体(pSJF2载体，kim Is.Biosic Biochem.2002,66(5):1148-51)，经T4连接酶连接重组，获得能在大肠杆菌中高效表达的质粒sdAb-pSJF2，并进行基因序列测定以确定其序列的正确性。
(1)获得CEACAM6的VHH目的基因的PCR扩增条件，50μl PCR体系扩增，PCR反应条件：先94℃，3分钟，然后进行94℃，30秒；72℃，45秒，52℃，30秒；共30循环；72℃，7分钟。5’引物—GAA GAAGAA GAC AA CAG GCC SAR GTG MAG CTG GWG GAK TCT；
3’引物—gaagatctccggatccTGAGGAGACGGTGACCTGGGT；
(2)目的基因和载体酶切，连接目的基因与载体，转化TG1，PCR鉴定含有目的片段的克隆，基因测序，获得基因序列正确的单域抗体表达质粒。
实施例4抗单域抗体的表达和纯化
将实施例3所述的含有质粒sdAb-pSJF2的菌种接种在含氨基苄青霉素的LB培养板上，37℃过夜。挑选单个菌落接种于15ml的含氨基苄青霉素的LB培养液中，37℃，摇床培养过夜。转种10ml过夜培养物于1L含氨基苄青霉素的2YT培养液中，37℃摇床培养，240转/分，培养到OD值达0.4～0.6时，加入0.5～1.0mM IPTG，继续培养过夜。离心，收菌。加25％高渗蔗糖溶液，提取细胞周质中的可溶性表达的单域抗体，离心，收上清。经Ni+离子亲和层析获得纯度达90％以上的蛋白。图3为表达的部分CEACAM6抗单域抗体蛋白SDS-PAGE电泳结果，图4为表达的部分CEACAM6-sdAB经镍柱纯化后SDS-PAGE电泳结果。
实施例5纯化的CEACAM6单域抗体与CEACAM6抗原的结合试验(ELISA)
1.试验材料：可拆酶标板(Thermofisher公司)，CEACAM6抗原，Anti-Myc tagantibody-HRP(北京义翘神州生物技术有限公司)，TMB显色液(北京梅科万德，Cat：1001)，包被液pH 9.6，BSA(Sigma公司)。
2.试验方法
2.1分别包被Human CEACAM6 Protein，浓度2ug/ml，100ul/孔，4℃孵育过夜。
2.2加入2％脱脂牛奶PBS封闭，300ul/孔。37℃，孵育1.5h。
2.3稀释不同编号的CEACAM6单域抗体至终浓度为10.0ug/ml和1.0ug/ml，100ul/孔。
2.4稀释Anti-Myc tag antibody(HRP)(1:5000)，100ul/孔，37℃孵育1h。
2.5加入TMB显色液，100ul/孔，避光反应10min。
2.6加入50ul/孔2M H2SO4终止反应。
2.7在450nm波长测OD值。
3.试验结果
图5为纯化的CEACAM6单域抗体与人CEACAM6抗原特异性结合的活性试验结果。
实施例6抗CEACAM6单域抗体亲和力测定试验
1)样品制备抗原：Bio-CEACAM6用1×动态缓冲液(1×PBS，含0.05％Tween 20、0.1％BSA，pH 7.2)稀释至10μg/ml；
单域抗体：用1×kinetic缓冲液依次稀释为400nM、200nM、100nM、50nM、25nM、12.5nM、6.25nM；
2)样品测试
待测抗原通过SA sensor进行加载，抗原稀释5个稀释度，所有单域抗体的亲和力在50nm、20nm、10nm、1nm、0.1nm、0.01nm。部分单域抗体亲和力见表2，其亲和力范围见表2。
表2抗CEACAM6单域抗体亲和力测定结果
实施例7抗CEACAM6单域抗体人源化
人源化方法采用蛋白表面氨基酸人源化(Resurfacing)的方法及VHH人源化通用的抗原结合互补区移植法(CDR grafting to a universal framework)完成,并参照已申报专利(抗EGFR人源化的单域抗体、Fc融合蛋白、重链Fab蛋白及其应用，申请号：2019113490209)。
人源化步骤如下：对抗CEACAM6单域抗体NBC4、25和36同源建模，建模软件为Modeller9。参考可溶性好的人抗体DP-47和同源序列NBBcII10抗体的氨基酸序列，把抗CEACAM6单域抗体NBC4、25和36进行人源化。
人源化结果见表3。
表3 NBC4、25和36单域抗体人源化结果
*注释：X*：表示该氨基酸可能进行人源化变化位点。根据文献研究报道，超过80％，其免疫原性就接近人源抗体。
实施例8抗CEACAM6人源化单域抗体Fc融合蛋白的载体构建
(1)第一结构：sdAb1-Hinger-CH2-CH3(IgG1-Fc)。sdAb＝NBC4HM2或NBC25HM3或NBC36HM2。(2)构建步骤：NBC4HM2或NBC25HM3或NBC36HM2+人IgG1-Fc基因全合成，加入XhoI-EcoRI双酶切，将sdAb-Fc基因连接至p327.7表达载体(专利公布号CN 104195173 A)，并加入相应酶切位点和终止密码子,用XbaI-SalI双酶切，将另一sdAb-Fc基因连接至已含有sdAb-Fc(已XhoI-EcoRI双酶切连接)的p327.7表达载体中，最终使一个载体有2条sdAb-Fc序列。
本发明所提供的抗CEACAM6人源化单域抗体、Fc融合蛋白以及重链Fab蛋白的氨基酸和基因的序列表见4。
表4抗CEACAM6人源化单域抗体、Fc融合蛋白、重链Fab蛋白的序列表
实施例9抗CEACAM6人源化单域抗体Fc融合蛋白的表达与纯化
将表达载体NBC4HM2-p327.7或NBC25HM3-p327.7或NBC36HM2-p327.7分别转染CHO/K1细胞，用MSX筛选稳定的蛋白高表达细胞株，共筛选到3株稳定表达细胞株，用稳定表达细胞株，在500ml摇瓶中培养进行蛋白表达。
蛋白质纯化：将上述细胞表达上清用蛋白A株亲和层析纯化，纯化蛋白置换为柠檬酸(0.05％Tween80，pH6.2)缓冲液。抗CEACAM6人源化单域抗体Fc融合蛋白载体所表达纯化的蛋白见图6(3种CEACAM6人源化单域抗体表达纯化后，SDS-PAGE还原胶和非还原胶电泳结果)。
上述融合蛋白表达载体表达的蛋白理论推算值为：分别含有688、688和682个氨基酸；分子量(MW)，经Hinger二硫键连接分子量分别为7.664KD、7.704KD和7.569KD,等电点分别为(pI)为7.88、7.30和7.61，纯化后蛋白电泳SDS-PAGE还原后分子量约为38KD左右与理论推算值一致。抗CEACAM6人源化单域抗体融合蛋白的亲和力测定试验同上述实施例6，亲和力分析结果见表5。
表5抗CEACAM6人源化单域抗体融合蛋白与人CEACAM6的亲和力分析结果
实施例10放射性同位素标记CEACAM6人源化单域抗体融合蛋白试验
1.试验方法
(1)抗体DFO修饰：反应瓶中取1mL抗体溶液(上述三种融合蛋白中的一种2mg/ml)+1mL 0.5M NaHCO3/Na2CO3溶液，测pH值至碱性；37℃搅拌反应40min。PD10柱纯化。(2)抗体标记：取少许89Zr，加入2M Na2CO3溶液，调节pH中性；(3)抗体质控：玻璃纤维纸，展开剂；柠檬酸钠体系。抗体标记物在原点，游离89Zr在前沿。抗体修饰及89Zr标记路线图见图7。
2.试验结果
三种抗体结构的单域抗体-Fc融合蛋白标记同位素89Zr，在小鼠肿瘤动物模型体内重要器官和肿瘤部位组织的分布结果见表6、图8和图9。
表6给予89Zr-CEACAM6后各组织放射性物质摄取％ID值(mean±SD，n＝6)
试验结果表明，该单域抗体-Fc融合同位素标记后能很好的特异性靶向小鼠体内移植肿瘤(非小细胞肺癌、胰腺癌等)。
SEQUENCE LISTING
<110> 北京纽安博生物技术有限公司
<120> 抗CEACAM6单域抗体、人源化单域抗体及其Fc融合蛋白和应用
<130> BJ-3038-200709A
<160> 26
<170> PatentIn version 3.5
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<213> Artifical sequence
<400> 2
Gly Ser Ser Ser Arg Ser Gly Ser Phe Ser Tyr Leu Val
1 5 10
<210> 3
<211> 16
<212> PRT
<213> Artifical sequence
<400> 3
Ala Ala Gln Thr Ala Ile Arg Ala Gly Val Arg Asp Asp Tyr Asp Phe
1 5 10 15
<210> 4
<211> 7
<212> PRT
<213> Artifical sequence
<400> 4
Ala Arg Ala Gly Ile Met Gly
1 5
<210> 5
<211> 12
<212> PRT
<213> Artifical sequence
<400> 5
Ala Met Gly Arg Ser Gly Ala Ser Ala Tyr Tyr Gln
1 5 10
<210> 6
<211> 15
<212> PRT
<213> Artifical sequence
<400> 6
Ala Ala Gly Asn Pro Ile Ala Leu Thr Thr Ala Gln Tyr Tyr Asp
1 5 10 15
<210> 7
<211> 7
<212> PRT
<213> Artifical sequence
<400> 7
Phe Arg Ile Asn Asn Met Gly
1 5
<210> 8
<211> 14
<212> PRT
<213> Artifical sequence
<400> 8
Ala Ser Val Thr Pro Gly Arg Asn Thr Asn Tyr Ala Asp Ser
1 5 10
<210> 9
<211> 8
<212> PRT
<213> Artifical sequence
<400> 9
Ser Ala Tyr Gly Pro Ser Gly Ala
1 5
<210> 10
<211> 123
<212> PRT
<213> Artifical sequence
<400> 10
Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Gly Ser Ser Ser Arg Ser Gly Ser Phe Ser Tyr Leu Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Asp Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gln Thr Ala Ile Arg Ala Gly Val Arg Asp Asp Tyr Asp Phe
100 105 110
Trp Gly Gln Gly Ala Gln Val Thr Val Ser Ser
115 120
<210> 11
<211> 123
<212> PRT
<213> Artifical sequence
<400> 11
Gln Val Lys Leu Glu 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 Gln Thr Ala Arg Ala Gly
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Met Gly Arg Ser Gly Ala Ser Ala Tyr Tyr Gln Asp Ser Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Val Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr His Cys
85 90 95
Ala Ala Gly Asn Pro Ile Ala Leu Thr Thr Ala Gln Tyr Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 12
<211> 115
<212> PRT
<213> Artifical sequence
<400> 12
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Val Ser Gly Ile Asp Phe Arg Ile Asn
20 25 30
Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Gln Arg Glu Leu Val
35 40 45
Ala Ser Val Thr Pro Gly Arg Asn Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr Ser Cys Ser
85 90 95
Ala Tyr Gly Pro Ser Gly Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 13
<211> 369
<212> DNA
<213> Artifical sequence
<400> 13
caggtaaagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg caccttcagt aattatgcca tgggctggtt ccgcgaggct 120
ccagggaagg agcgtgagtt tgtcggatct agtagccgga gtggtagttt cagttacttg 180
gtagactccg tgaagggccg attcaccatc ttcagagaca acgacaagaa cacggtatat 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agcacagacg 300
gccatacggg ctggtgtacg tgacgactat gacttttggg gccagggagc ccaggtcacc 360
gtgtcctca 369
<210> 14
<211> 369
<212> DNA
<213> Artifical sequence
<400> 14
caggtaaagc tggaggagtc tgggggaggc ttggtgcagc ctgggggctc tctgagactc 60
tcctgtgcag cctctggaca gaccgccagg gccggcatca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgaatt tgtagcagct atgggacgga gtggtgctag cgcatattat 180
caagactccg tgcagggccg atttaccatc tccagagaca acgcccagaa cacggtgttt 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt atcactgtgc agcaggaaat 300
cccatagcgc taactactgc tcaatattat gactattggg gccaggggac ccaggtcacc 360
gtctcctca 369
<210> 15
<211> 345
<212> DNA
<213> Artifical sequence
<400> 15
caggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60
tcctgtgtag tctctggaat tgacttccgt atcaataata tgggctggta ccgccaggct 120
ccaggaacgc agcgcgagtt ggtcgcaagt gtgactccag gtcgtaatac aaactacgca 180
gactccgtga agggccgatt caccatctcc agagacaatg cccagaacac ggtgtatctg 240
caaatgaaca gtctgcaacc tgaagacacg gccgtctatt cgtgtagtgc atatggacca 300
tccggggcct attggggcca ggggacccag gtcaccgtct cctca 345
<210> 16
<211> 123
<212> PRT
<213> Artifical sequence
<400> 16
Glu Val Lys Leu Glu 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 Gln Thr Ala Arg Ala Gly
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Met Gly Arg Ser Gly Ala Ser Ala Tyr Tyr Gln Asp Ser Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Phe
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 Gly Asn Pro Ile Ala Leu Thr Thr Ala Gln Tyr Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 17
<211> 123
<212> PRT
<213> Artifical sequence
<400> 17
Glu Val Lys Leu Glu 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 Gln Thr Ala Arg Ala Gly
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Met Gly Arg Ser Gly Ala Ser Ala Tyr Tyr Gln Asp Ser Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Phe
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 Gly Asn Pro Ile Ala Leu Thr Thr Ala Gln Tyr Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 18
<211> 123
<212> PRT
<213> Artifical sequence
<400> 18
Gln Val Lys Leu Glu 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 Gln Thr Ala Arg Ala Gly
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Met Gly Arg Ser Gly Ala Ser Ala Tyr Tyr Gln Asp Ser Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Phe
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 Gly Asn Pro Ile Ala Leu Thr Thr Ala Gln Tyr Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 19
<211> 123
<212> PRT
<213> Artifical sequence
<400> 19
Glu Val Lys Leu Glu 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 Gln Thr Ala Arg Ala Gly
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Met Gly Arg Ser Gly Ala Ser Ala Tyr Tyr Gln Asp Ser Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Phe
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 Gly Asn Pro Ile Ala Leu Thr Thr Ala Gln Tyr Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 20
<211> 123
<212> PRT
<213> Artifical sequence
<400> 20
Glu Val Lys Leu Glu 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 Gln Thr Ala Arg Ala Gly
20 25 30
Ile Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Met Gly Arg Ser Gly Ala Ser Ala Tyr Tyr Gln Asp Ser Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Phe
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 Gly Asn Pro Ile Ala Leu Thr Thr Ala Gln Tyr Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 21
<211> 115
<212> PRT
<213> Artifical sequence
<400> 21
Gln 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 Val Ser Gly Ile Asp Phe Arg Ile Asn
20 25 30
Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Gly Leu Glu Leu Val
35 40 45
Ala Ser Val Thr Pro Gly Arg Asn Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser
85 90 95
Ala Tyr Gly Pro Ser Gly Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 22
<211> 115
<212> PRT
<213> Artifical sequence
<400> 22
Gln 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 Val Val Ser Gly Ile Asp Phe Arg Ile Asn
20 25 30
Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Gln Arg Glu Leu Val
35 40 45
Ala Ser Val Thr Pro Gly Arg Asn Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser
85 90 95
Ala Tyr Gly Pro Ser Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 23
<211> 349
<212> PRT
<213> Artifical sequence
<400> 23
Gln Val Lys Leu Glu 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 Gln Thr Ala Arg Ala Gly
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Met Gly Arg Ser Gly Ala Ser Ala Tyr Tyr Gln Asp Ser Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Phe
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 Gly Asn Pro Ile Ala Leu Thr Thr Ala Gln Tyr Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr
115 120 125
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
130 135 140
Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro
145 150 155 160
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
165 170 175
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
180 185 190
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
195 200 205
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
210 215 220
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
225 230 235 240
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
245 250 255
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
260 265 270
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
275 280 285
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
290 295 300
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
305 310 315 320
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His
325 330 335
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
340 345
<210> 24
<211> 2085
<212> DNA
<213> Artifical sequence
<400> 24
caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag catcttcagt aacgatgtca tgggctggta ccgccaggct 120
ccagggaagc agcgcgagtt ggtcgcgttt attactaacg gaggtgtcgc acactcgaaa 180
gaccccgaga agggacgatt caccatctcc agagacaatg gcaagaacac gctatatctg 240
cagatgaaca gcctcagagc tgacgacacg gccgtctatt actgtaatgt acggtcactc 300
gtcgaacgtt caagggagta ctggggccag gggacccagg tcaccgtctc ctcagataag 360
acccacactt gtcctccttg ccccgctcct gagctgctcg gcggcccatc tgtgtttctg 420
tttccaccaa agccaaagga tcagctcatg attagtagaa cacccgaggt gacatgcgtc 480
gtggttgatg tgagccacga agatcccgag gtcaagttta attggtacgt tgatggcgtg 540
gaggtgcaca acgcaaagac caagccacgc gaggagcagt acaatagcac ttaccgggtg 600
gtgagcgtgc tgaccgtgct gcaccaggat tggctcaatg gaaaggagta caagtgtaaa 660
gtctctaata aggctctgcc cgcacctatt gaaaaaacta ttagtaaggc taagggccag 720
cctagagagc cccaggtcta cacactgcca ccatctcgcg aggagatgac caagaatcag 780
gtgtccctga catgtctcgt caagggcttt taccctagcg atattgccgt cgagtgggag 840
agcaacggac agcctgagaa taattacaag acaaccccac ctgtgctcga ttccgacggc 900
agcttcttcc tgtactctaa gctcacagtc gataagtcca gatggcagca gggcaatgtg 960
ttttcttgta gtgtgctgca cgaggcactc cacaatcact acacacagaa gtccctgtcc 1020
ctcagtcccg gctaacaggt aaagctggag gagtctgggg gaggcttggt gcagcctggg 1080
ggctctctga gactctcctg tgcagcctct ggacagaccg ccagggccgg catcatgggc 1140
tggttccgcc aggctccagg gaagggactt gaatttgtag cagctatggg acggagtggt 1200
gctagcgcat attatcaaga ctccgtgcag ggccgattta ccatctccag agacaactcc 1260
cagaacacgc tgtttctgca aatgaacagc ctgagagctg aggacacggc cgtttattac 1320
tgtgcagcag gaaatcccat agcgctaact actgctcaat attatgacta ttggggccag 1380
gggaccctgg tcaccgtctc ctcagataag acccacactt gtcctccttg ccccgctcct 1440
gagctgctcg gcggcccatc tgtgtttctg tttccaccaa agccaaagga tcagctcatg 1500
attagtagaa cacccgaggt gacatgcgtc gtggttgatg tgagccacga agatcccgag 1560
gtcaagttta attggtacgt tgatggcgtg gaggtgcaca acgcaaagac caagccacgc 1620
gaggagcagt acaatagcac ttaccgggtg gtgagcgtgc tgaccgtgct gcaccaggat 1680
tggctcaatg gaaaggagta caagtgtaaa gtctctaata aggctctgcc cgcacctatt 1740
gaaaaaacta ttagtaaggc taagggccag cctagagagc cccaggtcta cacactgcca 1800
ccatctcgcg aggagatgac caagaatcag gtgtccctga catgtctcgt caagggcttt 1860
taccctagcg atattgccgt cgagtgggag agcaacggac agcctgagaa taattacaag 1920
acaaccccac ctgtgctcga ttccgacggc agcttcttcc tgtactctaa gctcacagtc 1980
gataagtcca gatggcagca gggcaatgtg ttttcttgta gtgtgctgca cgaggcactc 2040
cacaatcact acacacagaa gtccctgtcc ctcagtcccg gctaa 2085
<210> 25
<211> 341
<212> PRT
<213> Artifical sequence
<400> 25
Gln 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 Val Val Ser Gly Ile Asp Phe Arg Ile Asn
20 25 30
Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Gln Arg Glu Leu Val
35 40 45
Ala Ser Val Thr Pro Gly Arg Asn Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser
85 90 95
Ala Tyr Gly Pro Ser Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
115 120 125
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
130 135 140
Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
145 150 155 160
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
165 170 175
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
180 185 190
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
195 200 205
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
210 215 220
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
225 230 235 240
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
245 250 255
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
260 265 270
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
275 280 285
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
290 295 300
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
305 310 315 320
Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
325 330 335
Ser Leu Ser Pro Gly
340
<210> 26
<211> 1026
<212> DNA
<213> Artifical sequence
<400> 26
caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgtag tctctggaat tgacttccgt atcaataata tgggctggta ccgccaggct 120
ccaggaacgc agcgcgagtt ggtcgcaagt gtgactccag gtcgtaatac aaactacgca 180
gactccgtga agggccgatt caccatctcc agagacaatg cccagaacac gctgtatctg 240
caaatgaaca gtctgcgagc tgaagacacg gccgtctatt actgtagtgc atatggacca 300
tccggggcct attggggcca ggggaccctg gtcaccgtct cctcagataa gacccacact 360
tgtcctcctt gccccgctcc tgagctgctc ggcggcccat ctgtgtttct gtttccacca 420
aagccaaagg atcagctcat gattagtaga acacccgagg tgacatgcgt cgtggttgat 480
gtgagccacg aagatcccga ggtcaagttt aattggtacg ttgatggcgt ggaggtgcac 540
aacgcaaaga ccaagccacg cgaggagcag tacaatagca cttaccgggt ggtgagcgtg 600
ctgaccgtgc tgcaccagga ttggctcaat ggaaaggagt acaagtgtaa agtctctaat 660
aaggctctgc ccgcacctat tgaaaaaact attagtaagg ctaagggcca gcctagagag 720
ccccaggtct acacactgcc accatctcgc gaggagatga ccaagaatca ggtgtccctg 780
acatgtctcg tcaagggctt ttaccctagc gatattgccg tcgagtggga gagcaacgga 840
cagcctgaga ataattacaa gacaacccca cctgtgctcg attccgacgg cagcttcttc 900
ctgtactcta agctcacagt cgataagtcc agatggcagc agggcaatgt gttttcttgt 960
agtgtgctgc acgaggcact ccacaatcac tacacacaga agtccctgtc cctcagtccc 1020
ggctaa 1026
Claims (10)
1.一组抗CEACAM6的单域抗体，所述单域抗体均由框架区和3个互补决定区组成，其特征在于，所述单域抗体选自NBC10、NBC25或NBC36中的任何一种；其中，单域抗体NBC10的3个互补决定区的氨基酸序列分别为SEQ ID No.1、SEQ ID No.2和SEQ ID No.3所示；单域抗体NBC25的3个互补决定区的氨基酸序列分别为SEQ ID No.4、SEQ ID No.5和SEQ ID No.6所示；单域抗体NBC36的3个互补决定区的氨基酸序列分别为SEQ ID No.7、SEQ ID No.8和SEQID No.9所示；
或者将上述所示的任何一种氨基酸序列中的一个或多个氨基酸进行缺失、取代、插入和/或添加所得到的蛋白突变体，该蛋白突变体与突变前的蛋白具有相同的功能；或者与上述所示的任何一种氨基酸序列至少有90％以上同一性的氨基酸序列。
2.按照权利要求1所述的单域抗体，其特征在于，单域抗体NBC10的氨基酸序列为SEQID No.10所示，单域抗体NBC25的氨基酸序列为SEQ ID No.11所示，单域抗体NBC36的氨基酸序列为SEQ ID No.12所示；
或者将上述所示的任何一种氨基酸序列中的一个或多个氨基酸进行缺失、取代、插入和/或添加所得到的蛋白突变体，该蛋白突变体与突变前的蛋白具有相同的功能；或者与上述所示的任何一种氨基酸序列至少有90％以上同一性的氨基酸序列。
3.权利要求1或2任何一项所述单域抗体的编码基因；优选的，所述单域抗体NBC10的编码基因的核苷酸序列为SEQ ID No.13所示，所述单域抗体NBC25的编码基因的核苷酸序列为SEQ ID No.14所示，所述单域抗体NBC36的编码基因的核苷酸序列为SEQ ID No.15所示
或者与上述所示的任何一种多核苷酸序列的互补序列在严谨杂交条件能够进行杂交的多核苷酸序列；或者与上述所示的任何一种的多核苷酸序列至少有90％以上同一性的多核苷酸序列。
4.一种重组表达载体，其特征在于，所述重组表达载体包含权利要求3所述的编码基因中的一个或多个。
5.人源化后的抗CEACAM6的单域抗体，其特征在于，将单域抗体NBC25进行了人源化改造得到了5个人源化的抗体NBC25HM1、NBC25HM2、NBC25HM3、NBC25HM4和NBC25HM5，其氨基酸序列分别为SEQ ID No.16、SEQ ID No.17、SEQ ID No.18、SEQ ID No.19和SEQ ID No.20所示；将单域抗体NBC36进行了人源化改造得到了2个人源化的抗体NBC36HM1和NBC36HM2，其氨基酸序列分别为SEQ ID No.21和SEQ ID No.22所示。
6.一种融合蛋白，其特征在于，将权利要求1或2任何一种的单域抗体或权利要求5所述的人源化后的抗CEACAM6的单域抗体与IgG-Fc构建得到的融合蛋白；优选的，所述Fc基因序列是来源于IgG，IgA，IgM的Fc基因序列或来源于IgG1，IgG2，IgG3或IgG4；所述的IgG优选是人IgG及其IgG1、2、3、4的亚类、人IgM、人IgA或其它动物免疫球蛋白的Fc片段。
7.按照权利要求6所述的融合蛋白，其特征在于，其氨基酸序列为SEQ ID No.23所示，其编码基因的核苷酸序列为SEQ ID No.24所示；或者其氨基酸序列为SEQ ID No.25所示，其编码基因的核苷酸序列为SEQ ID No.26所示。
8.一种缀合物，其特征在于，将权利要求1、2或5所述的单域抗体与细胞毒性剂、酶相、放射性同位素或化学发光化合物中的一种或多种相偶联得到缀合物。
9.权利要求权利要求1、2或5所述的单域抗体、权利要求3所述的编码基因、权利要求6或7所述的融合蛋白以及权利要求8所述的缀合物在制备检测或诊断与CEACAM6有关的药物或试剂中的用途。
10.权利要求权利要求1、2或5所述的单域抗体、权利要求3所述的编码基因、权利要求6或7所述的融合蛋白以及权利要求8所述的缀合物制备用于治疗CEACAM6表达异常相关疾病的药物中的应用；优选的，所述的CEACAM6表达异常相关疾病包括非小细胞肺癌、胰腺癌、乳腺癌或卵巢癌。
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