抗体可变区稳定性评价及其初步应用

摘要

Objective To study the intrinsic relationships between the binding energy of the antibody light and heavy chains and the conformational characteristics , physical and chemical properties , and to establish a corresponding mathemat-ical model and evaluate the thermal stability of the antibody molecules , which contribute to the antibody design , optimiza-tion and affinity maturation .Methods Based on bioinformatics and computational biology methods , the antibody′s structur-al information with the crystal diffraction data was analyzed .The conformational character of the variable domain of the antibody was studied using distance geometry and computer graphics technology .With the aid of the intermolecular hydrogen bond formation theory and the reaction free energy theory , the dynamic structure and energy characteristics be-tween the heavy and light chain variable regions of the antibody were studied .Furthermore , using nonlinear fitting and regression analysis, a mathematical model was set up .Results According to simulation and statistic analysis , there was a linear relationship between the binding energy and the number of the intermolecular hydrogen bonding , Van der Waals interaction of the heavy and light chains of the antibody .There was polynomial correlation between the binding energy and the physicochemical properties of the antibody .Using the frequency of amino acid position and the established model , the humanized anti-ricin antibody , which could not obtain the stable engineering cell line , was evaluated and optimized .The stable engineering cell line of the humanized anti-ricin antibody was obtained in the experiment .Conclusion The self structure of the antibody variable region ( conformation and physicochemical properties ) has much effect on its stability . The antibody stability can be improved by structural optimization .%目的：通过研究抗体轻、重链间的结合能与其构象特征、理化性质间的内在关系，建立相应的数学模型，合理评价抗体分子的热稳定性，为抗体分子的设计、合理改造及亲和力成熟奠定基础。方法采用生物信息学和计算生物学相关方法对已有晶体结构的抗体结构信息进行分析，通过距离几何学、计算机图形学技术对抗体可变区的构象特征进行研究；基于分子间氢键形成理论、反应自由能理论，从热力学、动力学对抗体分子轻、重链可变区相互作用的动态结构及能量特征进行探讨；借助统计学原理、非线性拟合、回归分析等分析手段对抗体轻、重链作用能与其构象特征、理化性质建立相关性分析。结果通过分子模拟与统计学分析，抗体的轻、重链可变区结合能与其轻、重链间的氢键形成数目、范德华作用均存在线性相关性；与抗体理化性质存在基于多项式的非线性相关性。基于关键位置氨基酸的使用频率以及建立的分析模型，针对无法获得稳定工程细胞株的抗蓖麻毒素人源化功能抗体进行合理的评价及优化，借助理论指导，获得抗蓖麻毒素人源化抗体稳定的工程细胞株。结论抗体可变区的自身结构（构象、理化性质）对其稳定性有很大影响，可以通过抗体结构优化来提高抗体的稳定性。