Gupta Amita, Chaudhary Vijay K, Bhat Rajiv
Department of Biochemistry, University of Delhi South Campus, New Delhi, India.
MAbs. 2009 May-Jun;1(3):268-80. doi: 10.4161/mabs.1.3.8231.
We have earlier described a haemagglutination-based assay for on-site detection of antibodies to HIV using whole blood. The reagent in this assay comprises of monovalent Fab fragment of an anti-human RBC antibody fused to immunodominant antigens of HIV-1 and HIV-2. In the present work, we describe a rational and systematic method for directed evolution of scFv and Fab antihuman RBC antibody fragments. Based on homology modeling and germline sequence alignments of antibodies, target residues in the anti-RBC MAb B6 sequence were identified for mutagenesis.A combinatorial library of 107 clones was constructed and subjected to selection on whole RBC under competitive binding conditions to identify several phage-displayed B6 scFv clones with improved binding as determined in an agglutination assay.Selected VL and VH sequences were shuffled to generate a second generation phage-displayed Fab library which on panning yielded Fab clones with several fold better binding than wild type. The mutants with better binding also displayed more Fab molecules per phage particle indicating improved in vivo folding which was also confirmed by their increased periplasmic secretion compared to the wild type. The mutant Fab molecules also showed superior characteristics in large scale production by in vitro folding of LC and Fd. The biophysical measurements involving thermal and chemical denaturation and renaturation kinetics clearly showed that two of the mutant Fab molecules possessed significantly improved characteristics as compared to the wild type B6 Fab.Structural modelling revealed that B6 Fab mutants had increased hydrogen bonding resulting in increased stability. Our approach provides a novel and useful strategy to obtain recombinant antibodies with improved characteristics.
我们之前描述了一种基于血凝反应的检测方法，用于使用全血现场检测HIV抗体。该检测方法中的试剂由与HIV-1和HIV-2的免疫显性抗原融合的抗人红细胞抗体的单价Fab片段组成。在本研究中，我们描述了一种合理且系统的方法，用于对单链抗体片段（scFv）和Fab抗人红细胞抗体片段进行定向进化。基于抗体的同源建模和种系序列比对，确定了抗红细胞单克隆抗体B6序列中的目标残基用于诱变。构建了一个包含107个克隆的组合文库，并在竞争结合条件下对全红细胞进行筛选，以鉴定出几个在凝集试验中显示出结合能力提高的噬菌体展示B6 scFv克隆。将选定的轻链（VL）和重链（VH）序列进行改组，以产生第二代噬菌体展示Fab文库，该文库在淘选后产生的Fab克隆的结合能力比野生型高几倍。结合能力更好的突变体每个噬菌体颗粒还展示出更多的Fab分子，表明体内折叠得到改善，这也通过与野生型相比其周质分泌增加得到证实。突变的Fab分子在通过轻链（LC）和重链可变区（Fd）的体外折叠进行大规模生产时也表现出优异的特性。涉及热变性、化学变性和复性动力学的生物物理测量清楚地表明，与野生型B6 Fab相比，其中两个突变Fab分子具有显著改善的特性。结构建模显示，B6 Fab突变体的氢键增加，导致稳定性提高。我们的方法提供了一种新颖且有用的策略，以获得具有改善特性的重组抗体。
