Effective Optimization of Antibody Affinity by Phage Display Integrated with High-Throughput DNA Synthesis and Sequencing Technologies.
Effective Optimization of Antibody Affinity by Phage Display Integrated with High-Throughput DNA Synthesis and Sequencing Technologies.
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噬菌体展示结合高通量 DNA 合成和测序技术有效优化抗体亲和力
DOI：
10.1371/journal.pone.0129125
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Hong J
中科院分区：
文献类型：
--
作者：
Hu D;Hu S;Wan W;Xu M;Du R;Zhao W;Gao X;Liu J;Liu H;Hong J
噬菌体展示技术已广泛用于抗体亲和力成熟数十年。有限的文库序列多样性以及过度的冗余和用于候选物鉴定的耗费人力的程序是该技术被广泛采用的两个主要障碍。我们在此描述了一种新的文库生成和筛选方法来解决这些问题。该方法开始于人源化抗ErbB2抗体HuA21的多个互补决定区（CDR）的靶向多样化，采用小扰动诱变策略。选择三个简并密码子NWG、NWC和NSG的组合用于氨基酸饱和诱变，而不引入半胱氨酸和终止残基。总共在两个微芯片上合成了7，749个简并寡核苷酸，并释放以构建具有4 X IO6个DNA序列的5个单链抗体片段（scFv）基因文库。使用Illumina平台对噬菌体文库和选择的噬菌体文库进行深度测序，允许深入评估CDR序列和氨基酸取代中的富集景观。使用NGS分析根据其高频率鉴定有效的候选物，绕过对靶结合克隆的初步筛选的需要。此外，通过重组来自四个CDR的10个最丰富的变体构建并筛选随后的文库，并且获得具有158倍增加的亲和力（Kd = 25.5pM）的突变体。这些结果表明，开发的方法用于优化其他抗体和生物分子的结合特性的潜在应用。
Phage display technology has been widely used for antibody affinity maturation for decades. The limited library sequence diversity together with excessive redundancy and labour-consuming procedure for candidate identification are two major obstacles to widespread adoption of this technology. We hereby describe a novel library generation and screening approach to address the problems. The approach started with the targeted diversification of multiple complementarity determining regions (CDRs) of a humanized anti-ErbB2 antibody, HuA21, with a small perturbation mutagenesis strategy. A combination of three degenerate codons, NWG, NWC, and NSG, were chosen for amino acid saturation mutagenesis without introducing cysteine and stop residues. In total, 7,749 degenerate oligonucleotides were synthesized on two microchips and released to construct five single-chain antibody fragment (scFv) gene libraries with 4 x 106 DNA sequences. Deep sequencing of the unselected and selected phage libraries using the Illumina platform allowed for an in-depth evaluation of the enrichment landscapes in CDR sequences and amino acid substitutions. Potent candidates were identified according to their high frequencies using NGS analysis, by-passing the need for the primary screening of target-binding clones. Furthermore, a subsequent library by recombination of the 10 most abundant variants from four CDRs was constructed and screened, and a mutant with 158-fold increased affinity (Kd = 25.5 pM) was obtained. These results suggest the potential application of the developed methodology for optimizing the binding properties of other antibodies and biomolecules.
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绕过体外筛选——下一代测序技术应用于抗体展示和计算机候选物选择。
影响因子：
14.9
作者：
Ravn, U.;Gueneau, F.;Baerlocher, L.;Osteras, M.;Desmurs, M.;Malinge, P.;Magistrelli, G.;Farinelli, L.;Kosco-Vilbois, M. H.;Fischer, N.
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影响因子：
5.3
作者：
González-Muñoz A;Bokma E;O'Shea D;Minton K;Strain M;Vousden K;Rossant C;Jermutus L;Minter R
通讯作者：
Minter R
影响因子：
14.9
作者：
Borovkov AY;Loskutov AV;Robida MD;Day KM;Cano JA;Le Olson T;Patel H;Brown K;Hunter PD;Sykes KF
通讯作者：
Sykes KF
DOI：
10.1073/pnas.1215549109
发表时间：
2012-11-06
影响因子：
11.1
作者：
Larman, H. Benjamin;Xu, George Jing;Elledge, Stephen J.
通讯作者：
Elledge, Stephen J.