Department of Medical Biotechnology and Molecular Science, North Khorasan University of Medical Science, Bojnurd, Iran.
Biotechnology Research Center, Pasture Institute of Iran, Tehran, Iran.
Biochem Biophys Res Commun. 2018 Nov 30;506(3):653-659. doi: 10.1016/j.bbrc.2018.10.127. Epub 2018 Oct 26.
Antibody engineering is now a noteworthy area in biopharmaceuticals as the next generation of marketed antibodies is engineered antibodies such as affinity- or stability-improved antibodies, fragmented or fused antibodies, antibody drug conjugates (ADCs), and PEGylated antibody fragments. In the current study, affinity enhancement of Nb against PlGF was performed by an in silico affinity maturation and molecular dynamics (MD) simulation. First, 300 single-point mutants were designed by identifying the residues involved in interaction with PlGF and different energy distributions. An energy based screening was performed to select best single-point mutants. Additionally, one variant containing two mutations was designed based on the selected single-point mutants. Finally, mutants-PlGF complexes were analyzed in details by all atom MD simulation. Trajectory analysis revealed that in both single (L112H, S31D, A97K, and R45E) and double (S31D & R45E) mutants, the free binding energies and the stability of complexes were significantly improved. The highest increment in affinity was observed for S31D mutant due to substantial increase in polar and electrostatic interactions. The secondary structure of Nb was intact in all variants and a shrinkage of PlGF over Nb was observed in all mutant-PlGF complexes during simulation. In addition, contact area and hydrogen-bond analysis as well as distance measurement in mutants-PlGF complexes also confirmed the affinity enhancement of variants relative to the native form. Our study showed that ligand-based affinity improvement could be considered as a promising approach for designing high affinity fragmented antibodies.
抗体工程现在是生物制药领域的一个重要领域，因为下一代上市的抗体是工程抗体，如亲和力或稳定性改善的抗体、片段化或融合抗体、抗体药物偶联物 (ADC) 和聚乙二醇化抗体片段。在本研究中，通过计算亲和力成熟和分子动力学 (MD) 模拟来提高 Nb 对 PlGF 的亲和力。首先，通过鉴定与 PlGF 相互作用的残基和不同的能量分布来设计 300 个单点突变体。进行基于能量的筛选以选择最佳的单点突变体。此外，根据选定的单点突变体设计了一个包含两个突变的变体。最后，通过全原子 MD 模拟详细分析突变体-PlGF 复合物。轨迹分析表明，在单个（L112H、S31D、A97K 和 R45E）和双（S31D 和 R45E）突变体中，自由结合能和复合物的稳定性都得到了显著提高。由于极性和静电相互作用的大幅增加，S31D 突变体的亲和力增加最高。在所有变体中，Nb 的二级结构保持完整，并且在模拟过程中观察到 PlGF 在所有突变体-PlGF 复合物上覆盖 Nb 的收缩。此外，突变体-PlGF 复合物中的接触面积和氢键分析以及距离测量也证实了变体相对于天然形式的亲和力增强。我们的研究表明，基于配体的亲和力改善可以被认为是设计高亲和力片段化抗体的一种有前途的方法。
