Department of Molecular Medicine, Beckman Research Institute at City of Hope, Duarte, California 91010, United States.
ACS Chem Biol. 2011 Sep 16;6(9):962-70. doi: 10.1021/cb200176v. Epub 2011 Jul 26.
The clinical application of siRNA is limited largely by the lack of efficient, cell-specific delivery systems. Antibodies are attractive delivery vehicles for targeted therapy due to their high specificity. In this study we describe the use of a humanized monoclonal antibody (mAb), hu3S193, against Lewis-Y (Le(y)), as a delivery vehicle for STAT3 siRNA. This mAb is rapidly internalized into Le(y)-expressing cancer cells via antigen recognition, and when coupled to STAT3 siRNA, a potentially powerful molecularly targeted delivery agent is created. Selective silencing of STAT3 is associated with tumor suppression. Two hu3S193 based siRNA delivery systems using STAT3 siRNA as a prototype were developed and tested in Le(y)-positive cancer cells: (a) a covalent construct based on a reductive disulfide linker that is expected to undergo cleavage within cells and (b) a noncovalent construct based on (d-arginine)(9) (9r) modified hu3S193. Le(y)-specific binding and internalization of both the covalent and noncovalent constructs were confirmed by flow cytometry and confocal microscopy. Both the covalent and the noncovalent system led to efficient STAT3 silencing in Le(y)-positive cancer cells (A431) but not in Le(y)-negative cancer cells (MDA-MB-435). The covalent construct, however, required co-treatment with reagents such as chloroquine or 9r that facilitate the escape of the siRNA from endosomes to achieve significant gene silencing. The 9r modified noncovalent construct induced ∼70% STAT3 knockdown at submicromolar siRNA concentrations when used at an optimal vehicle-to-siRNA ratio of 5:1. The STAT3 knockdown also led to ∼50% inhibition of cell proliferation of Le(y)-positive cells. Noncovalent linked STAT3 siRNA-hu3S193 has great promise for targeted knockdown of STAT3 in tumor cells.
siRNA 的临床应用在很大程度上受到缺乏高效、细胞特异性递送系统的限制。抗体由于其高度特异性，是靶向治疗的有吸引力的递送载体。在这项研究中，我们描述了使用针对 Lewis-Y（Le(y)）的人源化单克隆抗体（mAb）hu3S193 作为 STAT3 siRNA 的递送载体。这种 mAb 通过抗原识别快速内化到表达 Le(y)的癌细胞中，当与 STAT3 siRNA 偶联时，就会产生一种潜在的强大的分子靶向递送剂。STAT3 的选择性沉默与肿瘤抑制有关。我们开发并测试了两种基于 hu3S193 的 STAT3 siRNA 递送系统，将 STAT3 siRNA 用作原型，用于 Le(y)阳性癌细胞：(a) 基于还原二硫键连接子的共价构建体，预计在细胞内发生裂解；(b) 基于（d-精氨酸）（9）（9r）修饰的 hu3S193 的非共价构建体。通过流式细胞术和共聚焦显微镜证实了两种共价和非共价构建体的 Le(y)特异性结合和内化。两种共价和非共价系统都能有效地使 Le(y)阳性癌细胞（A431）中的 STAT3 沉默，但不能使 Le(y)阴性癌细胞（MDA-MB-435）中的 STAT3 沉默。然而，共价构建体需要与氯喹或 9r 等试剂共同处理，以促进 siRNA 从内体逃逸，从而实现显著的基因沉默。当使用最佳的载体与 siRNA 的比例为 5:1 时，9r 修饰的非共价构建体诱导的 STAT3 敲低约为 70%，在亚毫摩尔 siRNA 浓度下。STAT3 的敲低也导致 Le(y)阳性细胞的细胞增殖抑制约 50%。非共价连接的 STAT3 siRNA-hu3S193 具有在肿瘤细胞中靶向敲低 STAT3 的巨大潜力。
