基于计算模拟的抗Her2抗体A21的亲和力及人源化改造
发布时间：2018-08-05 20:15
【摘要】：嵌合抗体chA21是由本实验室构建的一株抗p185/Her2的工程化人-鼠嵌合抗体,对p185/Her2高表达的肿瘤具有明显的抑制活性。与另一株已用于临床的抗p185/Her2抗体药物Herceptin相比,chA21在抗原亲和力和抗体免疫原性方面还有一些不足。为了使其更适用于肿瘤治疗,我们基于蛋白结构模拟对chA21进行了亲和力改造和人源化改造的研究。 我们通过计算设计点突变的方法对A21的亲和力进行了改造。首先我们通过点突变扫描,精确化了A21的抗原识别表位,并建立了新的抗体互补决定区(CDR)的分类方法,用这种新方法对抗体A21的各CDR区内的参与抗原识别的位点进行了预测。在这些分析结果基础上,通过计算机分子对接模拟,更精细地揭示了A21和Her2的相互作用模式,并通过计算预测,设计了改造抗体A21的抗原亲和力的点突变方案并进行了初步实验验证。另外,我们结合分子对接模拟和之前的活性测定结果,从结构角度阐述了A2l的识别表位与其肿瘤抑制机制的相关性。 为了降低嵌合抗体chA21的免疫原性,我们通过CDR移植的方法对其鼠源可变区进行了人源化改造。基于A21可变区的序列和结构信息,选择人源化抗体huCC49作为其人源化模板进行抗原结合区移植,基于移植之后人化抗体的模拟结构,对一些重要的框架区残基进行突变方向的筛选,选择更符合抗体结构稳定需要的突变方向,得到A2l的人源化变体H1、H1-1、H1-2的序列。在293T真核表达系统中对Fc融合的人源化变体进行表达,亲和力分析结果表明,与嵌合抗体A21相比,设计的A21人源化抗体H1、H1-1、H1-2与抗原的亲和力相近,并已经达到了完全的人源化,具有更好的临床应用前景。
[Abstract]:Chimeric antibody chA21 is an engineering human-mouse chimeric antibody against p185/Her2 constructed in our laboratory. It has obvious inhibitory activity against tumor with high expression of p185/Her2. Compared with another strain of Herceptin, which has been used in clinic, Herceptin has some deficiencies in antigen affinity and immunogenicity. In order to make it more suitable for tumor therapy, we studied the modification of affinity and humanization of chA21 based on protein structure simulation. We modified the affinity of A 21 by calculating the point mutation. First of all, the antigen recognition epitopes of A21 were accurately identified by point mutation scanning, and a new classification method of (CDR) in the complementary determinant region of antibody was established. The sites involved in antigen recognition in each CDR region of antibody A21 were predicted by this new method. On the basis of these results, the interaction model between A21 and Her2 is revealed in more detail by computer molecular docking simulation, and calculated and predicted. A point mutation method was designed to modify the antigen affinity of antibody A 21, and a preliminary experiment was carried out to verify it. In addition, based on the results of molecular docking simulation and previous activity determination, the relationship between the recognition epitopes of A21 and its tumor inhibition mechanism was discussed from the structural point of view. In order to reduce the immunogenicity of chimeric antibody chA21, we humanized the murine variable region by CDR transplantation. Based on the sequence and structure information of A21 variable region, humanized antibody huCC49 was selected as its humanized template for antigen binding region transplantation, based on the simulated structure of humanized antibody after transplantation. The mutation direction of some important frame residues was screened, and the mutation direction which was more suitable for the stability of antibody structure was selected, and the sequence of the human variant H1H1-1H1-2 of A21 was obtained. The human strain of FC fusion was expressed in 293T eukaryotic expression system. The results of affinity analysis showed that compared with chimeric antibody A21, the affinity of the designed human antibody H1H1-1H1-2 to antigen was similar to that of antigen, and it had been completely humanized. It has better prospect of clinical application.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
本文编号：2166895
[Abstract]:Chimeric antibody chA21 is an engineering human-mouse chimeric antibody against p185/Her2 constructed in our laboratory. It has obvious inhibitory activity against tumor with high expression of p185/Her2. Compared with another strain of Herceptin, which has been used in clinic, Herceptin has some deficiencies in antigen affinity and immunogenicity. In order to make it more suitable for tumor therapy, we studied the modification of affinity and humanization of chA21 based on protein structure simulation. We modified the affinity of A 21 by calculating the point mutation. First of all, the antigen recognition epitopes of A21 were accurately identified by point mutation scanning, and a new classification method of (CDR) in the complementary determinant region of antibody was established. The sites involved in antigen recognition in each CDR region of antibody A21 were predicted by this new method. On the basis of these results, the interaction model between A21 and Her2 is revealed in more detail by computer molecular docking simulation, and calculated and predicted. A point mutation method was designed to modify the antigen affinity of antibody A 21, and a preliminary experiment was carried out to verify it. In addition, based on the results of molecular docking simulation and previous activity determination, the relationship between the recognition epitopes of A21 and its tumor inhibition mechanism was discussed from the structural point of view. In order to reduce the immunogenicity of chimeric antibody chA21, we humanized the murine variable region by CDR transplantation. Based on the sequence and structure information of A21 variable region, humanized antibody huCC49 was selected as its humanized template for antigen binding region transplantation, based on the simulated structure of humanized antibody after transplantation. The mutation direction of some important frame residues was screened, and the mutation direction which was more suitable for the stability of antibody structure was selected, and the sequence of the human variant H1H1-1H1-2 of A21 was obtained. The human strain of FC fusion was expressed in 293T eukaryotic expression system. The results of affinity analysis showed that compared with chimeric antibody A21, the affinity of the designed human antibody H1H1-1H1-2 to antigen was similar to that of antigen, and it had been completely humanized. It has better prospect of clinical application.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
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