Gong Rui, Chen Weizao, Dimitrov Dimiter S
Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
Methods Mol Biol. 2012;899:85-102. doi: 10.1007/978-1-61779-921-1_6.
Most of the FDA-approved therapeutic monoclonal antibodies are full-size IgG molecules with a molecular weight of about 150 kDa. A major problem for such large molecules is their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules (e.g., on the HIV envelope glycoprotein) which are fully accessible only by molecules of smaller size. Therefore, much work especially during the last decade has been aimed at developing novel scaffolds of much smaller size and high stability. Immunoglobulin-based scaffolds including Fab (∼50 kD), ScFv (∼30 kD), and VH domain (termed domain antibody, dAb) (∼15 kD) have been well established. Recently, a new scaffold based on human IgG1 CH2 domain (∼15 kD) was also proposed (termed nanoantibody, nAb). Binders based on a CH2 scaffold could also confer some effector functions. Here, we describe the design, expression, purification, and characterization of engineered CH2 and VH domains.
大多数经美国食品药品监督管理局（FDA）批准的治疗性单克隆抗体是分子量约为150 kDa的全尺寸IgG分子。这类大分子的一个主要问题是它们难以穿透组织（如实体瘤），并且与某些分子表面区域（如HIV包膜糖蛋白上的区域）的结合能力较差或不存在，而这些区域只有较小尺寸的分子才能完全接近。因此，特别是在过去十年中，大量工作致力于开发尺寸更小、稳定性更高的新型支架。基于免疫球蛋白的支架，包括Fab（约50 kD）、ScFv（约30 kD）和VH结构域（称为结构域抗体，dAb）（约15 kD）已经得到了充分的研究。最近，还提出了一种基于人IgG1 CH2结构域（约15 kD）的新型支架（称为纳米抗体，nAb）。基于CH2支架的结合物也可以赋予一些效应功能。在此，我们描述了工程化CH2和VH结构域的设计、表达、纯化和表征。
