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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Affinity Maturation Of Antibodies Assisted By In Silico Modeling
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Affinity Maturation Of Antibodies Assisted By In Silico Modeling

机译：计算机模拟辅助抗体的亲和力成熟

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摘要

Rational engineering methods can be applied with reasonable success to optimize physicochemical characteristics of proteins, in particular, antibodies. Here, we describe a combined CDR3 walking randomization and rational design-based approach to enhance the affinity of the human anti-gastrin TA4 scFv. The application of this methodology to TA4 scFv, displaying only a weak overall affinity forgastrin17 (K_D = 6 μM), resulted in a set of nine affinity-matured scFv variants with near-nanomolar affinity (K_D = 13.2 nM for scFv TA4.112). First, CDR-H3 and CDR-L3 randomization resulted in three scFvs with an overall affinity improvement of 15- to 35-fold over the parental. Then, the modeling of two scFv constructs selected from the previous step (TA4.11 and TA4.13) was followed by a combination of manual and molecular dynamics-based docking of gastrin17 into the respective binding sites, analysis of apparent packing defects, and selection of residues for mutagenesis through phage display. Nine scFv mutants were obtained from the second maturation step. A final 454-fold improvement in affinity compared with TA4 was obtained. These scFvs showed an enhanced potency to inhibit gastrin-induced proliferation in Colo 320 WT and BxPc3 tumoral cells. In conclusion, we propose a structure-based rational method to accelerate the development of affinity-matured antibody constructs with enhanced potential for therapeutic use.
机译：可以合理合理地应用合理的工程方法来优化蛋白质（尤其是抗体）的理化特性。在这里，我们描述了结合CDR3步行随机化和基于合理设计的方法来增强人类抗胃泌素TA4 scFv的亲和力。将该方法应用于TA4 scFv时，其对胃泌素17的总体亲和力很弱（K_D = 6μM），从而产生了九个亲和力成熟的scFv变异体，具有近纳摩尔亲和力（scFv TA4.112的K_D = 13.2 nM） 。首先，CDR-H3和CDR-L3随机化产生了三个scFv，与亲本相比，总体亲和力提高了15到35倍。然后，对从上一步骤（TA4.11和TA4.13）中选择的两个scFv构建体进行建模，然后将基于手动和分子动力学的gastrin17停靠到各个结合位点，分析明显的包装缺陷和通过噬菌体展示选择用于诱变的残基。从第二个成熟步骤中获得了9个scFv突变体。与TA4相比，亲和力最终提高了454倍。这些scFvs在Colo 320 WT和BxPc3肿瘤细胞中抑制胃泌素诱导的增殖的能力增强。总之，我们提出了一种基于结构的合理方法，以加快具有增强治疗潜力的亲和力成熟的抗体构建体的开发。

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