Institute of Immunology, University of Heidelberg, Heidelberg, Germany.
Curr Cancer Drug Targets. 2010 Dec;10(8):922-31. doi: 10.2174/156800910793357952.
The efficacy of cancer-immunotherapy with complement-activating monoclonal antibodies is limited by over-expression of one or more membrane-bound complement regulatory proteins (mCRPs: CD46, CD55, CD59) on the surface of neoplastic cells. In this study we designed small interfering RNAs (siRNAs) for posttranscriptional gene knock down of CD46, CD55 and CD59 aiming at to sensitize tumor cells to complement attack and thereby to better exploit complement for tumor cell destruction. Tumor cell lines of different origin, such as Du145 (prostate), BT474 (breast) and K562 (erythroleukemia) were selected for the study. FACS-analysis demonstrated that siRNA anti-CD46(301) reduced CD46 protein expression up to 80%, siRNA anti-CD55(255) diminished CD55 protein expression up to 49%, and CD59 protein expression was inhibited up to 82% by siRNA anti-CD59(1339). Time course experiments revealed a long-lasting silencing effect with >50% complement regulator inhibition up to day 13. Upon mCRP knock down, complement-dependent cytotoxicity (CDC) was augmented by 20-30% for CD46, by up to 24% for CD55 and by up to 55% for CD59. The combined inhibition of all three inhibitors further enhanced CDC by up to 66%. Dependent on the cell line, CD46 and CD55 downregulation increased significantly C3 ospsonization, which is known to support cell-mediated defense mechanisms. mCRP blocking antibodies were only partly able to further augment the tumor cells' susceptibility to complement lysis. Thus, siRNA-induced inhibition of complement regulator expression clearly sensitizes malignant cells to complement attack and, if specifically targeted to the tumor, appears suited as adjuvant to improve antibody-based cancer immunotherapy.
癌症免疫疗法的疗效受到肿瘤细胞表面过度表达一种或多种膜结合补体调控蛋白（mCRP：CD46、CD55、CD59）的限制。在这项研究中，我们设计了小干扰 RNA（siRNA），用于 CD46、CD55 和 CD59 的转录后基因敲低，旨在使肿瘤细胞对补体攻击敏感，从而更好地利用补体破坏肿瘤细胞。选择不同来源的肿瘤细胞系，如 Du145（前列腺）、BT474（乳腺）和 K562（红白血病）进行研究。FACS 分析表明，siRNA 抗 CD46(301)使 CD46 蛋白表达降低了 80%，siRNA 抗 CD55(255)使 CD55 蛋白表达降低了 49%，siRNA 抗 CD59(1339)使 CD59 蛋白表达抑制了 82%。时程实验表明，抑制效果持续时间长，超过 50%的补体调控蛋白抑制作用可持续至第 13 天。mCRP 敲低后，CD46 的补体依赖性细胞毒性（CDC）增加了 20-30%，CD55 增加了 24%，CD59 增加了 55%。三种抑制剂的联合抑制进一步使 CDC 增强了高达 66%。依赖于细胞系，CD46 和 CD55 的下调显著增加了 C3 调理作用，这已知可支持细胞介导的防御机制。mCRP 阻断抗体只能部分增强肿瘤细胞对补体溶解的敏感性。因此，siRNA 诱导的补体调控蛋白表达抑制可明显增强恶性细胞对补体攻击的敏感性，如果特异性靶向肿瘤，可作为改善基于抗体的癌症免疫疗法的辅助手段。
