Single domain intracellular antibodies:: A minimal fragment for direct in vivo selection of antigen-specific intrabodies
Single domain intracellular antibodies:: A minimal fragment for direct in vivo selection of antigen-specific intrabodies
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DOI：
10.1016/s0022-2836(03)00836-2
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发表时间：
2003-08-29
影响因子：
5.6
通讯作者：
Rabbitts, TH
中科院分区：
文献类型：
--
作者：
Tanaka, T;Lobato, MN;Rabbitts, TH
在靶标验证和治疗应用中，主要需要能够干扰细胞内蛋白质功能的分子。细胞内抗体（intrabodies）可以结合细胞中的特异性靶标，但目前难以分离intrabodies。胞内抗体通常是包含免疫球蛋白重链（VH）和轻链（VL）的可变结构域的单链Fv片段。我们现在证明，单VH结构域具有优异的细胞内溶解性、稳定性和在高等生物体细胞内的表达，并且可以在体内表现出特异性抗原识别。我们已经使用这种细胞内单可变结构域（IDab）格式，基于先前表征的胞内抗体共有支架，以产生用于直接体内筛选的多种胞内抗体文库。使用两种不同的抗原分离IDab，并且分离的IDab的亲和力在20 nM和200 nM之间。此外，选择与RAS蛋白结合的IDabs可以抑制NIH 3T3细胞的RAS依赖性致癌转化。因此，IDab形式对于体内胞内抗体使用是理想的。这种胞内抗体的方法消除了对噬菌体抗体文库的需要，避免了体外产生抗原的要求，并允许在体内直接选择胞内抗体。(C)2003爱思唯尔有限公司。保留所有权利。
There is a major need in target validation and therapeutic applications for molecules that can interfere with protein function inside cells. Intracellular antibodies (intrabodies) can bind to specific targets in cells but isolation of intrabodies is currently difficult. Intrabodies are normally single chain Fv fragments comprising variable domains of the immunoglobulin heavy (VH) and light chains (VL). We now demonstrate that single VH domains have excellent intracellular properties of solubility; stability and expression within the cells of higher organisms and can exhibit specific antigen recognition in vivo. We have used this intracellular single variable domain (IDab) format, based on a previously characterised intrabody consensus scaffold, to generate diverse intrabody libraries for direct in vivo screening. IDabs were isolated using two distinct antigens and affinities of isolated IDabs ranged between 20 nM and 200 nM. Moreover, IDabs selected for binding to the RAS protein could inhibit RAS-dependent oncogenic transformation of NIH3T3 cells. The IDab format is therefore ideal for in vivo intrabody use. This approach to intrabodies obviates the need for phage antibody libraries, avoids the requirement for production of antigen in vitro and allows for direct selection of intrabodies in vivo. (C) 2003 Elsevier Ltd. All rights reserved.