HAb18G/CD147及其单抗的分子模建对接和抗体人源化设计的研究
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摘要
随着计算机辅助分子模拟技术的蓬勃发展,计算生物学越来越多地应用于生命科学各个领域,包括蛋白质结构分析、预测和分子对接的理论方法和应用软件。这些方法和工具用于抗体结构和抗原-抗体相互作用的研究当中,将为抗体工程相关的研究和应用提供了重要的理论指导和实验所需的基础数据。
本研究旨在:1)用公共访问的生物信息学工具和数据库,建立一种针对鼠源性和人源性抗体可变区结构预测和模拟的方法,并转化为可用的软件工具。同时用该软件对HAb18G/CD147单体HAb18进行结构模建;2)通过计算机辅助分子对接技术研究HAb18G/CD147分子与抗体HAb18的结合模式,确定抗原-抗体相互作用的关键氨基酸残基。3)在分析抗体空间结构特点和氨基酸残基间相互作用基础上,利用计算机辅助分子设计和表面残基替换法进行鼠抗体人源化设计。
研究方法是:1)抗体结构模建:从PDB(Protein Data Bank)数据库中筛选抗体的结构信息,构建本地抗体结构库,使用Java语言编写程序实现目标抗体同本地抗体结构库的序列比对,为目标抗体搜索同源模板,最后由Modeller 9v1完成抗体三维结构预测;2)抗原表位预测:以折叠识别为模建方法、DOPE(Discrete Optimized Protein Energy)得分为筛选标准、AMBER6为优化工具,构建HAb18G/CD147胞外两个结构域,通过Insight II实现HAb18G/CD147同HAb18抗体分子对接并分析相互作用位点;3)基于表面重塑方法的抗体人源化设计是在抗体同源模建的基础上,利用序列分析和结构分析确定鼠源抗体可变区外露的非人样差异残基,参考分子内和分子间氢键相互作用并选定将要突变的残基位点。
研究结果:1)成功建立了一套抗体结构预测和模建的方法,构建了抗体HAb18可变区的三维模型。开发出一个简单易用、具有图形界面和交互功能的抗体结构预测软件AbModeller,能在RMSD(Root Mean Square Deviation)等于2?的范围内准确的构建抗体可变区结构;2)模拟了HAb18G/CD147分子胞外段同其抗体的对接模型,确定了组成表位的关键氨基酸残基,为实验研究提供了理论参考;3)完成了HAb18可变区的人源化设计,并将确定的候选位点分为三类,以便在实验中依次突变,寻找既降低鼠源抗体免疫原性又保持生物学功能的平衡点。
本文通过建立抗原、抗体结构预测和分子对接的方法和工具有力地支持了抗体生物学的各项研究工作,为单抗人源化设计、抗原-抗体分子表位识别和基于抗原-抗体相互作用位点结构的药物设计提供了理论依据和参考,并在一定程度上改变了传统抗体研究的模式。
Along with vigorous development of computer assisted molecule simulation methodology, Computational biology is increasingly used in all fields of life sciences. Under such situation, many theoretical methods and application software have presented in protein structure analysis and prediction and molecule docking. The application of these methods and tools in the study about antibody structure and interaction of antigen-antibody will provide strong theoretical guidance and foundation data, which are required by experiments, for antibody engineering-related research.
The first purpose of our study was to establish a method of 3-D structure prediction and simulation of mouse and human antibody variable regions based on public-accessible bioinformatics tools and databases. Furthermore, put methods into available software tools. Using this software, an anti-HAB18G/CD147 monoclonal antibody is modeled. The second purpose was to verify the epitope and the key residues by analyzed the complex model of HAb18G/CD147 extracellular portion and its MAb based on computer assisted molecule docking. The third purpose was to perform a design of humanizing murine antibody with the method of residue replacement based on analyzing of antibody spatial structure and interaction among residues by means of computer assisted molecule design.
There were three parts in our study: 1) Antibody structure modeling. A local database of antibody 3-D structure was constructed by extracting antibody resource from PDB. Sequence alignment and homology template search against the local database for query antibody was performed by a Java program and modeling was realized by Modeller 9v1. 2) Epitope prediction. The model of extracellular portion of HAb18G/CD147 molecule was constructed by a serial of steps that included fold recognition, DOPE evaluation and model refinement using AMBER6. The docking of HAb18G/CD147 extracellular portion and its MAb was performed by InsightII. 3) Humanization design of MAb HAb18 based on variable domain resurfacing. Identification of the surface exposed non-human like residues of MAb variable region and calculation of inter-molecular and intra-molecular hydrogen bond interaction by sequence and structure analysis in terms of the homology modeled variable region of mAb determined the residues that need to be mutated to their human counterpart.
In this study, we proposed a method of predicting antibody 3-D structure and developed a software tool named by AbModeller using to construct antibody model that is easy to be used and contain user interface and interactive function. In 2? of RMSD, AbModeller can predict accurately antibody spatial structure. Moreover, to simulate the complex model of HAb18G/CD147 extracellular portion and its MAb by molecule docking and identify the key residues of epitope that can be a theoretical evidence for experiment. Finally, the antibody variable domain resurfacing method was applied to the humanization of the monoclonal antibodies HAb18 against human hepatoma cell. The candidate mutation sites were put into three categories in experiment for maintaining an appropriate balance between the biological function and reduced immunogenicity.
In conclusion, the methods and software of modeling and docking of antigen-antibody can support effectively the research of antibody biology and provide theoretical guidance for humanization and epitope identification and drug design based on the sites of antigen-antibody interaction.
本研究旨在:1)用公共访问的生物信息学工具和数据库,建立一种针对鼠源性和人源性抗体可变区结构预测和模拟的方法,并转化为可用的软件工具。同时用该软件对HAb18G/CD147单体HAb18进行结构模建;2)通过计算机辅助分子对接技术研究HAb18G/CD147分子与抗体HAb18的结合模式,确定抗原-抗体相互作用的关键氨基酸残基。3)在分析抗体空间结构特点和氨基酸残基间相互作用基础上,利用计算机辅助分子设计和表面残基替换法进行鼠抗体人源化设计。
研究方法是:1)抗体结构模建:从PDB(Protein Data Bank)数据库中筛选抗体的结构信息,构建本地抗体结构库,使用Java语言编写程序实现目标抗体同本地抗体结构库的序列比对,为目标抗体搜索同源模板,最后由Modeller 9v1完成抗体三维结构预测;2)抗原表位预测:以折叠识别为模建方法、DOPE(Discrete Optimized Protein Energy)得分为筛选标准、AMBER6为优化工具,构建HAb18G/CD147胞外两个结构域,通过Insight II实现HAb18G/CD147同HAb18抗体分子对接并分析相互作用位点;3)基于表面重塑方法的抗体人源化设计是在抗体同源模建的基础上,利用序列分析和结构分析确定鼠源抗体可变区外露的非人样差异残基,参考分子内和分子间氢键相互作用并选定将要突变的残基位点。
研究结果:1)成功建立了一套抗体结构预测和模建的方法,构建了抗体HAb18可变区的三维模型。开发出一个简单易用、具有图形界面和交互功能的抗体结构预测软件AbModeller,能在RMSD(Root Mean Square Deviation)等于2?的范围内准确的构建抗体可变区结构;2)模拟了HAb18G/CD147分子胞外段同其抗体的对接模型,确定了组成表位的关键氨基酸残基,为实验研究提供了理论参考;3)完成了HAb18可变区的人源化设计,并将确定的候选位点分为三类,以便在实验中依次突变,寻找既降低鼠源抗体免疫原性又保持生物学功能的平衡点。
本文通过建立抗原、抗体结构预测和分子对接的方法和工具有力地支持了抗体生物学的各项研究工作,为单抗人源化设计、抗原-抗体分子表位识别和基于抗原-抗体相互作用位点结构的药物设计提供了理论依据和参考,并在一定程度上改变了传统抗体研究的模式。
Along with vigorous development of computer assisted molecule simulation methodology, Computational biology is increasingly used in all fields of life sciences. Under such situation, many theoretical methods and application software have presented in protein structure analysis and prediction and molecule docking. The application of these methods and tools in the study about antibody structure and interaction of antigen-antibody will provide strong theoretical guidance and foundation data, which are required by experiments, for antibody engineering-related research.
The first purpose of our study was to establish a method of 3-D structure prediction and simulation of mouse and human antibody variable regions based on public-accessible bioinformatics tools and databases. Furthermore, put methods into available software tools. Using this software, an anti-HAB18G/CD147 monoclonal antibody is modeled. The second purpose was to verify the epitope and the key residues by analyzed the complex model of HAb18G/CD147 extracellular portion and its MAb based on computer assisted molecule docking. The third purpose was to perform a design of humanizing murine antibody with the method of residue replacement based on analyzing of antibody spatial structure and interaction among residues by means of computer assisted molecule design.
There were three parts in our study: 1) Antibody structure modeling. A local database of antibody 3-D structure was constructed by extracting antibody resource from PDB. Sequence alignment and homology template search against the local database for query antibody was performed by a Java program and modeling was realized by Modeller 9v1. 2) Epitope prediction. The model of extracellular portion of HAb18G/CD147 molecule was constructed by a serial of steps that included fold recognition, DOPE evaluation and model refinement using AMBER6. The docking of HAb18G/CD147 extracellular portion and its MAb was performed by InsightII. 3) Humanization design of MAb HAb18 based on variable domain resurfacing. Identification of the surface exposed non-human like residues of MAb variable region and calculation of inter-molecular and intra-molecular hydrogen bond interaction by sequence and structure analysis in terms of the homology modeled variable region of mAb determined the residues that need to be mutated to their human counterpart.
In this study, we proposed a method of predicting antibody 3-D structure and developed a software tool named by AbModeller using to construct antibody model that is easy to be used and contain user interface and interactive function. In 2? of RMSD, AbModeller can predict accurately antibody spatial structure. Moreover, to simulate the complex model of HAb18G/CD147 extracellular portion and its MAb by molecule docking and identify the key residues of epitope that can be a theoretical evidence for experiment. Finally, the antibody variable domain resurfacing method was applied to the humanization of the monoclonal antibodies HAb18 against human hepatoma cell. The candidate mutation sites were put into three categories in experiment for maintaining an appropriate balance between the biological function and reduced immunogenicity.
In conclusion, the methods and software of modeling and docking of antigen-antibody can support effectively the research of antibody biology and provide theoretical guidance for humanization and epitope identification and drug design based on the sites of antigen-antibody interaction.
引文
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