Department of Biology, University of Rome Tor Vergata, Rome, Italy.
Department of Infectious Diseases, Italian National Institute of Health, Rome, Italy.
Biomed Res Int. 2018 May 20;2018:6583852. doi: 10.1155/2018/6583852. eCollection 2018.
In Human Papillomaviruses- (HPV-) associated carcinogenesis, continuous expression of the E6 oncoprotein supports its value as a potential target for the development of diagnostics and therapeutics for HPV cancer. We previously reported that the I7 single-chain antibody fragment (scFv) specific for HPV16 E6, expressed as an intrabody by retroviral system, could inhibit significantly the growth of cervical cancer cells and was even able to reduce tumor development in experimental HPV-related cancer models. Nevertheless, for the development of therapeutic tools to be employed in humans, it is important to achieve maximum safety guarantee, which can be provided by the protein format. In the current study, two anti-16E6 scFvs derived from I7 were expressed in and purified in soluble form by affinity chromatography. Specificity, sensitivity and stability in physiologic environment of the purified scFvs were demonstrated by binding studies using recombinant 16E6 as an antigen. The scFvs functionality was confirmed by immunofluorescence in cervical cancer cells, where the scFvs were able to recognize the nuclear E6. Furthermore, an antiproliferative activity of the scFvI7nuc delivered in protein format to HPV16-positive cell lines was observed. Our results demonstrate that functional anti-16E6 scFvs can be produced in , suggesting that such purified antibodies could be used in the diagnosis and treatment of HPV-induced malignancies.
在人乳头瘤病毒（HPV）相关的致癌过程中，E6 癌蛋白的持续表达使其成为开发 HPV 癌症诊断和治疗方法的潜在靶点。我们之前曾报道过，针对 HPV16 E6 的 I7 单链抗体片段（scFv），通过逆转录病毒系统表达为内抗体，能够显著抑制宫颈癌细胞的生长，甚至能够减少实验性 HPV 相关癌症模型中的肿瘤发展。然而，为了开发可供人类使用的治疗工具，确保最大安全性是非常重要的，而这可以通过蛋白质形式提供。在本研究中，从 I7 中表达了两种针对 16E6 的抗 scFv，并通过亲和层析以可溶形式进行了纯化。通过使用重组 16E6 作为抗原进行结合研究，证明了纯化的 scFv 的特异性、敏感性和在生理环境中的稳定性。通过免疫荧光在宫颈癌细胞中证实了 scFv 的功能，scFv 能够识别核 E6。此外，还观察到以蛋白质形式递送至 HPV16 阳性细胞系的 scFvI7nuc 的抗增殖活性。我们的研究结果表明，可以在 中生产功能性抗 16E6 scFv，这表明这种纯化的抗体可用于 HPV 诱导的恶性肿瘤的诊断和治疗。
