HPV Unit, UOSD Tumor Immunology and Immunotherapy, IRCCS Regina Elena National Cancer Institute, 00144, Rome, Italy.
Department of Biology, University of Rome Tor Vergata, 00133, Rome, Italy.
J Exp Clin Cancer Res. 2021 Jan 23;40(1):37. doi: 10.1186/s13046-021-01841-w.
The oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells.
Envisioning clinical application of the best characterized anti-HPV16 E6 and -HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed.
We showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway.
Based on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.
高危型人乳头瘤病毒 16 型（HPV16）的致癌活性完全依赖于病毒感染期间产生的 E6 和 E7 病毒癌蛋白。癌蛋白通过促进增殖、抑制凋亡和阻止上皮分化来干扰细胞内稳态，使受感染的细胞向肿瘤进展。E6/E7 的表达与细胞转化之间的因果关系允许通过抑制两种癌蛋白的活性来抑制致癌过程。我们之前开发并鉴定了一些针对 HPV16 E6 和 E7 蛋白的单链抗体（scFv），并在体外和体内证实了它们的抗肿瘤活性，包括对 HPV16 阳性细胞肿瘤进展的保护作用。
为了将最好的抗 HPV16 E6 和抗 HPV16 E7 scFv 应用于临床，我们验证了它们在已植入肿瘤的治疗环境中的活性。表达具有核靶向序列的抗 HPV16 E6 scFvI7 或具有内质网靶向序列的抗 HPV16 E7 scFv43M2 的重组质粒通过注射和电穿孔递送至使用 C57/BL6 小鼠的三种不同的临床前模型中，并研究了它们对肿瘤生长的影响。在第一个模型中，使用 HPV16+TC-1Luc 细胞在小鼠中植入肿瘤，并通过荧光素酶活性测量肿瘤生长；在第二个模型中，使用四倍数量的 TC-1 细胞获得生长更快的肿瘤；在第三个模型中，使用 HPV16+C3 细胞在小鼠中生成肿瘤。为了突出 scFv 的可能作用机制，对肿瘤切片进行了 H&E 和 caspase-3 染色。
我们表明，在三种实验模型中，测试的两种抗 HPV16 E6 和 HPV16 E7 scFv 均有效延迟肿瘤进展，并且它们的抗肿瘤活性似乎依赖于诱导肿瘤细胞走向凋亡途径。
基于我们的研究，已经鉴定出两种 scFv，它们可能代表一种安全有效的 HPV16 相关病变治疗方法。scFv 有效性的机制似乎是通过凋亡导致细胞死亡。此外，还证实了电穿孔作为一种允许用于人类治疗的方法，将 scFv 递送至肿瘤的有效性。
