Grim J E, Siegal G P, Alvarez R D, Curiel D T
Gene Therapy Program, University of Alabama at Birmingham, Birmingham, Alabama, 35294, USA.
Biochem Biophys Res Commun. 1998 Sep 29;250(3):699-703. doi: 10.1006/bbrc.1998.9391.
The use of intracellular single chain antibodies has recently emerged as a highly efficient method of down-regulating or ablating protein expression. In this regard, we have demonstrated that a single chain antibody directed against the extracellular domain of the erbB-2 molecule causes a specific toxicity in erbB-2 positive tumor types. To further investigate the mechanism of this effect, we developed a second anti-erbB-2 sFv predicted to recognize an alternate extracellular epitope of the erbB-2 molecule. When produced as a secreted protein from the erbB-2 negative COS-1 cell line, this sFv binds specifically to erbB-2 positive cells, indicating that cellular machinery is able to produce a properly folded and functional sFv protein. However, by several assays, this sFv was shown to be unable to retain the erbB-2 protein within the ER. These negative results have implications for the evaluation and utilization of sFv knockout strategies in experimental contexts.
细胞内单链抗体的应用最近已成为一种高效下调或消除蛋白质表达的方法。在这方面，我们已经证明，一种针对erbB-2分子胞外结构域的单链抗体在erbB-2阳性肿瘤类型中会产生特异性毒性。为了进一步研究这种效应的机制，我们开发了第二种抗erbB-2单链抗体片段（sFv），预计它能识别erbB-2分子的另一个胞外表位。当从erbB-2阴性的COS-1细胞系作为分泌蛋白产生时，这种sFv能特异性结合erbB-2阳性细胞，表明细胞机制能够产生正确折叠且有功能的sFv蛋白。然而，通过多种检测方法发现，这种sFv无法将erbB-2蛋白保留在内质网中。这些阴性结果对于在实验环境中评估和利用sFv敲除策略具有重要意义。
