Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Institutes of Health, Frederick, Maryland, United States of America.
PLoS One. 2012;7(8):e42288. doi: 10.1371/journal.pone.0042288. Epub 2012 Aug 7.
Libraries based on an isolated human immunoglobulin G1 (IgG1) constant domain 2 (CH2) have been previously diversified by random mutagenesis. However, native isolated CH2 is not very stable and the generation of many mutations could lead to an increase in immunogenicity. Recently, we demonstrated that engineering an additional disulfide bond and removing seven N-terminal residues results in an engineered antibody domain (eAd) (m01s) with highly increased stability and enhanced binding to human neonatal Fc receptor (FcRn) (Gong et al, JBC, 2009 and 2011). We and others have also previously shown that grafting of the heavy chain complementarity region 3 (CDR-H3 (H3)) onto cognate positions of the variable domain leads to highly diversified libraries from which a number of binders to various antigens have been selected. However, grafting of H3s to non-cognate positions in constant domains results in additional residues at the junctions of H3s and the CH2 framework. Here we describe a new method based on multi-step PCR that allows the precise replacement of loop FG (no changes in its flanking sequences) by human H3s from another library. Using this method and limited mutagenesis of loops BC and DE we generated an eAd phage-displayed library. Panning of this library against an HIV-1 gp41 MPER peptide resulted in selection of a binder, m2a1, which neutralized HIV-1 isolates from different clades with modest activity and retained the m01s capability of binding to FcRn. This result provides a proof of concept that CH2-based antigen binders that also mimic to certain extent other functions of full-size antibodies (binding to FcRn) can be generated; we have previously hypothesized that such binders can be made and coined the term nanoantibodies (nAbs). Further studies in animal models and in humans will show how useful nAbs could be as therapeutics and diagnostics.
基于人免疫球蛋白 G1（IgG1）恒定域 2（CH2）的文库已经通过随机诱变进行了多样化。然而，天然分离的 CH2 不太稳定，并且产生许多突变可能会导致免疫原性增加。最近，我们证明，通过工程化额外的二硫键并去除七个 N 端残基，可以产生具有高度增加稳定性和增强与人新生儿 Fc 受体（FcRn）结合的工程抗体域（eAd）（m01s）（Gong 等人，JBC，2009 年和 2011 年）。我们和其他人以前还表明，将重链互补决定区 3（CDR-H3（H3））嫁接在可变域的同源位置会导致高度多样化的文库，从中选择了许多针对各种抗原的结合物。然而，将 H3 嫁接在恒定域的非同源位置会导致 H3 与 CH2 框架的连接处有额外的残基。在这里，我们描述了一种基于多步 PCR 的新方法，该方法允许精确地用来自另一个文库的人 H3 替换 FG 环（其侧翼序列不变）。使用这种方法和对 BC 和 DE 环的有限诱变，我们生成了一个 eAd 噬菌体展示文库。该文库针对 HIV-1 gp41 MPER 肽进行淘选，选择了一种结合物 m2a1，该结合物能够中和来自不同分支的 HIV-1 分离株，具有适度的活性，并保留了 m01s 与 FcRn 结合的能力。这一结果提供了一个概念验证，即也在某种程度上模拟全长抗体其他功能（与 FcRn 结合）的基于 CH2 的抗原结合物可以被生成；我们以前假设可以制造这种结合物，并将其命名为纳米抗体（nAbs）。在动物模型和人类中的进一步研究将表明 nAbs 作为治疗剂和诊断剂的有用性。
