利用导向选择及噬菌体抗体库技术制备人源性抗肝癌抗体
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摘要
原发性肝癌是我国常见恶性肿瘤之一。死亡率高，在恶性肿瘤死亡顺位中仅次于胃、食道而居第三位，在部分农村中则占第二位，仅次于胃癌。我国每年死于肝癌约13万人，占全世界肝癌死亡人数的45％。抗体工程的发展为临床肿瘤的诊断和治疗提供了新的途径，目前全世界约有80种治疗性单克隆抗体，许多单抗既可用作治疗剂，又可用作体内诊断剂。基于鼠源性抗肝癌单抗(McAb)HAb18的免疫诊断和治疗剂已进入临床试验。但鼠源性McAb注入人体会引起人抗鼠抗体(HAMA)反应，因而应用受到限制。重组DNA技术可以将动物抗体转变为人源性越来越高的抗体，即利用重组技术对鼠抗体基因进行改造，生产出天然不存在的人造抗体。目前已经利用人抗体恒定区与动物抗体可变区连接所构建的嵌合抗体，对特异性高，亲和力好的鼠McAb进行人源化改造。由于这两部分空间构象相对独立，保留了鼠单抗的可变区，特异性的抗源亲和力较高，同时其抗原性明显降低。但鼠源可变区的存在，仍可引起较强烈的HAMA反应。
目前已有多种途径进行鼠源性McAb的人源化改造，噬菌体展示抗体库技术的发展为人源抗体的制备提供了一个有力的技术平台。噬菌体展示技术是将外源蛋白分子展示于丝状噬粒表面的一种技术手段。近来，大容量的噬菌体抗体库已被建立，在体外不经免疫或者杂交瘤技术而得到人源性抗体，并易于制备稀有抗原的抗体、筛选全人源性抗体和高亲和力抗体。通过将所有抗体可变区基因克隆在噬菌体
草四军医，匕拳俘士拳位论义
中表达，利用不同的抗原筛选出携带特异抗体基因的克隆，从而获得
相应的特异性抗体。“导向选择”链更替技术可将鼠源性McAb改造
为与鼠McAb具备相似结合特性的完全人源化抗体。本实验利用抗肝
癌抗体HAbls嵌合轻链为模板，导向筛选抗体Fd片段;再以此为基础，
筛选出人源轻链基因，完成对HAbls的人源化改造。
第一部分:人杭体Fab片段基因扩增引物的优化和鉴定.构建噬菌体
抗体库，最重要的一步就是要尽量将所有的抗体基因扩增出来，提高
库容量，保持多样性。根据V-BASE提供的人抗体可变区胚系基因，
在其家族分类基础上，根据FRI区5’端保守序列重新分组，设计特异
性的5’端扩增引物。Fd3’端引物序列配对于Y链、p链的铰链区，轻
链3’端引物配对于轻链(K、入)恒定区3’端。同时对设计引物的匹
配情况进行系统分析，并应用PCR扩增和测序反应对其扩增效果进行
鉴定。在本实验中，优化设计一套人抗体Fab片段基因扩增引物。其
中，Fd链5’端扩增引物有5条，K轻链有6条5’端扩增引物，而入轻
链5’端扩增引物有10条。分析表明，人抗体可变区胚系基因与5’端
引物有较好的匹配率。与报道的建库常用引物比较，数目较少，匹配
率较高。PCR结果显示，全部的重链及轻链扩增引物对均能高效扩增出
特异性较高的目的片段。测序结果表明PCR扩增引物具有较好的亚类
和可变区家族分布。通过本部分工作，在人抗体可变区胚系基因家族
分类基础上，根据其5’端保守序列重新分组，优化设计特异性引物，
可以保证扩增效率，有利于大容量人源噬菌体Fab抗体库的构建和筛
选。
第二部分:噬菌体展示F ab抗体库构建和筛选条件的优化。自肝癌患
者外周血淋巴细胞扩增人重链Fd片段基因，分别克隆入含抗HAb 18G
嵌合轻链的展示载体pComb3和pC0mb3X，建立人一鼠杂合Fab噬菌
体抗体库(库容量均>lo7PFu)。并对抗体库的扩增和筛选条件进行优
化。实验表明，以XLI一Blue为宿主菌的抗体库，在37℃条件下进行扩
增挽救，存在明显的目的片段重组丢失现象。培养条件改为30℃时，
重组丢失现象受到明显的抑制。而以pC0mb3X为展示载体，在不同条
件下进行扩增挽救，重组丢失现象均能受到明显的抑制。利用GST融
合蛋白进行抗体库的筛选，由于广泛存在的交叉反应，影响筛选的效
布四月民医大拳俘士学位论文
里旦里里里里旦口里里国旦里旦旦鱼旦鱼国鱼鱼旦旦旦.口口鱼鱼鱼鱼旦鱼鱼坦鱼鱼里里里鱼鱼鱼鱼鱼鱼鱼鱼旦旦旦鱼鱼鱼鱼卫里鱼鱼鱼旦皿鱼皿皿皿旦旦鱼鱼里鱼鱼恤鱼鱼鱼口口..........
率。经过预筛选后，阳性克隆用噬菌体展示抗体进行ELISA检测，与
原核表达GST和无关蛋白存在明显交叉反应，并且存在明显的噬菌体
干扰。改用细菌诱导裂解上清进行筛选克隆ELISA鉴定，可以抑制交
叉反应，利于阳性克隆的鉴定。通过建立杂合Fab抗体库，并进行预
筛选，同时对扩增和筛选条件进行优化，建立一套有效的抗体库构建
和筛选方案。
第三部分:以嵌合杭体轻链为摸板导向筛选人源性杭肝癌杭体Fd片段.
利用建立的人一鼠杂合Fab噬菌体抗体库。以大肠杆菌表达的非融合
HAb18GE为抗原进行筛选，利用工PTG诱导表达的细菌裂解上清(pm
一Fab融合蛋白)进行克隆鉴定，并对筛选出的杂合抗体进行初步的功能
检测。利用非融合表达的HAb 18GE进行6轮筛选后，ELISA及流式
细胞仪检测，其中7个克隆呈特异性阳性反应，其中两个克隆信号较
强。通过本实验，以嵌合轻链为摸板，成功筛选到人源性抗体Fd片段。
第四部分:完全人源性杭肝病杭原HAb18G Fab抗体的筛选及初步功能
鉴定。将筛选得到?
Primary hepatic cell cancer (HCC) is one of the malignant tumors threatening the human. About 130 thousands patients dead of HCC every year in China,. The development of gene-engineering antibody provides a new method for the diagnosis and treatment of HCC. Now about 80 McAbs have enter the clinic trials. HAb18, the mouse anti-HCC McAb, have been complete the clinic trial with better prospect for the diagnosis and treatment. But as mouse McAb, HAMA(human anti-mouse antibody reaction) can be induced. Chimeric HAbl8-Fab with high humanization level still can induce significant HAMA.
The humanization of McAb can be completed by a set of techniques. Antibody library displayed on phage surface was a new technique developed in the last ten years. The technology of phage displaying antibody library allows much more rapid selection of specific antibodies from large libraries. The phage display antibody library provides a powerful methods for the production of gene-engineering McAb. Here the complete human anti-HAb18G Fabs were selected with guided-selection
and phage displaying antibody library techniques.
1.The optimization of the primers for the construction of human Fab
antibody library.
The construction of a large human phage antibody library with a good diversity is based on the amplification of all the human antibody genes.
According to the human germ-line V-genes in the V-BASE, the germ-line V-genes are divided into different groups. Based on these, the specific 5' primers are designed and optimized. All the germ-line V-genes are aligned with the 5' primers. With the match rate, the amplification efficiency is analyzed. The human Fd genes were amplified by RT-PCR from PBMC of hepatoma patients. Then with the primers, we amplified the genes of Fd and light chains, and the genes of PCR products were sequenced. In the study, we designed a set of specific 5 primers, including 5 primers for the Fd genes, 6 primers for the kappa chain and 10 primers for lamdda chain. All the primers have high match rate with the germ-line V-genes with a limited primer number, specially in the 3'regions of the primers. The results of PCR showed all primer-pairs could be used to amplify the interested antibody genes. Genes sequencing confirmed most interested genes could be amplified. The set of optimized family-specific primers can amplify the human genes of the Fd and light chains with a high efficiency. These will be helpful for the construction of human Fab antibody libraries. 2. The optimized strategy for the amplifying and screening of human-mouse phage Fab antibody library.
Based on the chimeric cFab-HAb18, the PCR products were cloned in the vectors pComb3 and pComb3X with the chimeric light chain anti-FLAb18G to construct the human-mouse Fab phage antibody libraries. In the study, the chimeric CL genes were paired as template with a repertoire of Fd chains, forming phage antibody library of human-mouse heterozygosis. With the GST fusion and no-fusion extracellular region of HAb18G, a cancer associated antigen, the target antibodies were selected. During the course, the strategy were optimized to increase the efficiency. When the host bacteria XLl-Blu was cultivated at 37 C, the clones could not cut out the correct fragments with the restriction endonucleases. It indicates the recombinant-deletion occurs. At 30C, about 17% of the clones had the phenomenon. GST-fusion HAb18GE had low efficacy because of the cross-action. In the study, the phage ELISA cannot be used the positive
clones selection because the serious cross-reaction. But the lyses supernatant after induced with IPTG could identify positive clones with ELISA. Through the construction of the HuMFab phage antibody library and the optimized screening strategy, we get the available procedures for subsequent research.
3. The selection of human anti-hepatoma Fd fragments guided with the chimeric light chain.
With the constructed human-mouse Fab phage library, HAbl8GE, extracellular region of HAb18G, were used as antigen to screening. The positive clones were ident
目前已有多种途径进行鼠源性McAb的人源化改造，噬菌体展示抗体库技术的发展为人源抗体的制备提供了一个有力的技术平台。噬菌体展示技术是将外源蛋白分子展示于丝状噬粒表面的一种技术手段。近来，大容量的噬菌体抗体库已被建立，在体外不经免疫或者杂交瘤技术而得到人源性抗体，并易于制备稀有抗原的抗体、筛选全人源性抗体和高亲和力抗体。通过将所有抗体可变区基因克隆在噬菌体
草四军医，匕拳俘士拳位论义
中表达，利用不同的抗原筛选出携带特异抗体基因的克隆，从而获得
相应的特异性抗体。“导向选择”链更替技术可将鼠源性McAb改造
为与鼠McAb具备相似结合特性的完全人源化抗体。本实验利用抗肝
癌抗体HAbls嵌合轻链为模板，导向筛选抗体Fd片段;再以此为基础，
筛选出人源轻链基因，完成对HAbls的人源化改造。
第一部分:人杭体Fab片段基因扩增引物的优化和鉴定.构建噬菌体
抗体库，最重要的一步就是要尽量将所有的抗体基因扩增出来，提高
库容量，保持多样性。根据V-BASE提供的人抗体可变区胚系基因，
在其家族分类基础上，根据FRI区5’端保守序列重新分组，设计特异
性的5’端扩增引物。Fd3’端引物序列配对于Y链、p链的铰链区，轻
链3’端引物配对于轻链(K、入)恒定区3’端。同时对设计引物的匹
配情况进行系统分析，并应用PCR扩增和测序反应对其扩增效果进行
鉴定。在本实验中，优化设计一套人抗体Fab片段基因扩增引物。其
中，Fd链5’端扩增引物有5条，K轻链有6条5’端扩增引物，而入轻
链5’端扩增引物有10条。分析表明，人抗体可变区胚系基因与5’端
引物有较好的匹配率。与报道的建库常用引物比较，数目较少，匹配
率较高。PCR结果显示，全部的重链及轻链扩增引物对均能高效扩增出
特异性较高的目的片段。测序结果表明PCR扩增引物具有较好的亚类
和可变区家族分布。通过本部分工作，在人抗体可变区胚系基因家族
分类基础上，根据其5’端保守序列重新分组，优化设计特异性引物，
可以保证扩增效率，有利于大容量人源噬菌体Fab抗体库的构建和筛
选。
第二部分:噬菌体展示F ab抗体库构建和筛选条件的优化。自肝癌患
者外周血淋巴细胞扩增人重链Fd片段基因，分别克隆入含抗HAb 18G
嵌合轻链的展示载体pComb3和pC0mb3X，建立人一鼠杂合Fab噬菌
体抗体库(库容量均>lo7PFu)。并对抗体库的扩增和筛选条件进行优
化。实验表明，以XLI一Blue为宿主菌的抗体库，在37℃条件下进行扩
增挽救，存在明显的目的片段重组丢失现象。培养条件改为30℃时，
重组丢失现象受到明显的抑制。而以pC0mb3X为展示载体，在不同条
件下进行扩增挽救，重组丢失现象均能受到明显的抑制。利用GST融
合蛋白进行抗体库的筛选，由于广泛存在的交叉反应，影响筛选的效
布四月民医大拳俘士学位论文
里旦里里里里旦口里里国旦里旦旦鱼旦鱼国鱼鱼旦旦旦.口口鱼鱼鱼鱼旦鱼鱼坦鱼鱼里里里鱼鱼鱼鱼鱼鱼鱼鱼旦旦旦鱼鱼鱼鱼卫里鱼鱼鱼旦皿鱼皿皿皿旦旦鱼鱼里鱼鱼恤鱼鱼鱼口口..........
率。经过预筛选后，阳性克隆用噬菌体展示抗体进行ELISA检测，与
原核表达GST和无关蛋白存在明显交叉反应，并且存在明显的噬菌体
干扰。改用细菌诱导裂解上清进行筛选克隆ELISA鉴定，可以抑制交
叉反应，利于阳性克隆的鉴定。通过建立杂合Fab抗体库，并进行预
筛选，同时对扩增和筛选条件进行优化，建立一套有效的抗体库构建
和筛选方案。
第三部分:以嵌合杭体轻链为摸板导向筛选人源性杭肝癌杭体Fd片段.
利用建立的人一鼠杂合Fab噬菌体抗体库。以大肠杆菌表达的非融合
HAb18GE为抗原进行筛选，利用工PTG诱导表达的细菌裂解上清(pm
一Fab融合蛋白)进行克隆鉴定，并对筛选出的杂合抗体进行初步的功能
检测。利用非融合表达的HAb 18GE进行6轮筛选后，ELISA及流式
细胞仪检测，其中7个克隆呈特异性阳性反应，其中两个克隆信号较
强。通过本实验，以嵌合轻链为摸板，成功筛选到人源性抗体Fd片段。
第四部分:完全人源性杭肝病杭原HAb18G Fab抗体的筛选及初步功能
鉴定。将筛选得到?
Primary hepatic cell cancer (HCC) is one of the malignant tumors threatening the human. About 130 thousands patients dead of HCC every year in China,. The development of gene-engineering antibody provides a new method for the diagnosis and treatment of HCC. Now about 80 McAbs have enter the clinic trials. HAb18, the mouse anti-HCC McAb, have been complete the clinic trial with better prospect for the diagnosis and treatment. But as mouse McAb, HAMA(human anti-mouse antibody reaction) can be induced. Chimeric HAbl8-Fab with high humanization level still can induce significant HAMA.
The humanization of McAb can be completed by a set of techniques. Antibody library displayed on phage surface was a new technique developed in the last ten years. The technology of phage displaying antibody library allows much more rapid selection of specific antibodies from large libraries. The phage display antibody library provides a powerful methods for the production of gene-engineering McAb. Here the complete human anti-HAb18G Fabs were selected with guided-selection
and phage displaying antibody library techniques.
1.The optimization of the primers for the construction of human Fab
antibody library.
The construction of a large human phage antibody library with a good diversity is based on the amplification of all the human antibody genes.
According to the human germ-line V-genes in the V-BASE, the germ-line V-genes are divided into different groups. Based on these, the specific 5' primers are designed and optimized. All the germ-line V-genes are aligned with the 5' primers. With the match rate, the amplification efficiency is analyzed. The human Fd genes were amplified by RT-PCR from PBMC of hepatoma patients. Then with the primers, we amplified the genes of Fd and light chains, and the genes of PCR products were sequenced. In the study, we designed a set of specific 5 primers, including 5 primers for the Fd genes, 6 primers for the kappa chain and 10 primers for lamdda chain. All the primers have high match rate with the germ-line V-genes with a limited primer number, specially in the 3'regions of the primers. The results of PCR showed all primer-pairs could be used to amplify the interested antibody genes. Genes sequencing confirmed most interested genes could be amplified. The set of optimized family-specific primers can amplify the human genes of the Fd and light chains with a high efficiency. These will be helpful for the construction of human Fab antibody libraries. 2. The optimized strategy for the amplifying and screening of human-mouse phage Fab antibody library.
Based on the chimeric cFab-HAb18, the PCR products were cloned in the vectors pComb3 and pComb3X with the chimeric light chain anti-FLAb18G to construct the human-mouse Fab phage antibody libraries. In the study, the chimeric CL genes were paired as template with a repertoire of Fd chains, forming phage antibody library of human-mouse heterozygosis. With the GST fusion and no-fusion extracellular region of HAb18G, a cancer associated antigen, the target antibodies were selected. During the course, the strategy were optimized to increase the efficiency. When the host bacteria XLl-Blu was cultivated at 37 C, the clones could not cut out the correct fragments with the restriction endonucleases. It indicates the recombinant-deletion occurs. At 30C, about 17% of the clones had the phenomenon. GST-fusion HAb18GE had low efficacy because of the cross-action. In the study, the phage ELISA cannot be used the positive
clones selection because the serious cross-reaction. But the lyses supernatant after induced with IPTG could identify positive clones with ELISA. Through the construction of the HuMFab phage antibody library and the optimized screening strategy, we get the available procedures for subsequent research.
3. The selection of human anti-hepatoma Fd fragments guided with the chimeric light chain.
With the constructed human-mouse Fab phage library, HAbl8GE, extracellular region of HAb18G, were used as antigen to screening. The positive clones were ident
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