Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
BMC Cancer. 2011 Jan 17;11:17. doi: 10.1186/1471-2407-11-17.
"High risk" human papillomavirus strains are the causative agents of the vast majority of carcinomas of the uterine cervix. In these tumors, the physical integration of the HPV genome is a frequent, though not invariable occurrence, but the constitutive expression of the E6 and E7 viral genes is always observed, suggesting key roles for the E6 and E7 oncoproteins in the process of malignant transformation. The "intracellular antibody" technology using recombinant antibodies in single-chain format offers the possibility of targeting a protein in its intracellular environment even at the level of definite domains thus representing a valuable strategy to "knock out" the function of specific proteins.
In this study, we investigate the in vitro activity of two single-chain antibody fragments directed against the "high-risk" HPV 16 E7 oncoprotein, scFv 43M2 and scFv 51. These scFvs were expressed by retroviral system in different cell compartments of the HPV16-positive SiHa cells, and cell proliferation was analyzed by Colony Formation Assay and EZ4U assay. The binding of these scFvs to E7, and their possible interference with the interaction between E7 and its main target, the tumor suppressor pRb protein, were then investigated by immunoassays, PepSet™ technology and Surface Plasmon Resonance.
The expression of the two scFvs in the nucleus and the endoplasmic reticulum of SiHa cells resulted in the selective growth inhibition of these cells. Analysis of binding showed that both scFvs bind E7 via distinct but overlapping epitopes not corresponding to the pRb binding site. Nevertheless, the binding of scFv 43M2 to E7 was inhibited by pRb in a non-competitive manner.
Based on the overall results, the observed inhibition of HPV-positive SiHa cells proliferation could be ascribed to an interaction between scFv and E7, involving non-pRb targets. The study paves the way for the employment of specific scFvs in immunotherapeutic approaches against the HPV-associated lesions.
“高危”型人乳头瘤病毒（HPV）是绝大多数宫颈癌的致病因素。在这些肿瘤中，HPV 基因组的物理整合是一种常见的，但并非不变的现象，但 HPV E6 和 E7 病毒基因的组成型表达总是观察到，这表明 E6 和 E7 癌蛋白在恶性转化过程中起着关键作用。使用单链形式的重组抗体的“细胞内抗体”技术提供了靶向其细胞内环境中蛋白质的可能性，甚至可以在特定结构域的水平上进行靶向，因此代表了“敲除”特定蛋白质功能的有价值策略。
在这项研究中，我们研究了两种针对“高危”型 HPV16 E7 癌蛋白的单链抗体片段 scFv43M2 和 scFv51 的体外活性。这些 scFv 通过逆转录病毒系统在 HPV16 阳性的 SiHa 细胞的不同细胞区室中表达，并通过集落形成测定和 EZ4U 测定分析细胞增殖。然后通过免疫测定、PepSet™技术和表面等离子体共振分析研究了这些 scFv 与 E7 的结合及其对 E7 与其主要靶标肿瘤抑制因子 pRb 蛋白之间相互作用的可能干扰。
两种 scFv 在 SiHa 细胞的核和内质网中的表达导致这些细胞的选择性生长抑制。结合分析表明，两种 scFv 通过不同但重叠的表位与 E7 结合，这些表位与 pRb 结合位点不对应。然而，scFv43M2 与 E7 的结合被 pRb 以非竞争性方式抑制。
基于总体结果，观察到 HPV 阳性 SiHa 细胞增殖的抑制可以归因于 scFv 与 E7 之间的相互作用，涉及非 pRb 靶标。该研究为针对 HPV 相关病变的特异性 scFv 免疫治疗方法的应用铺平了道路。
