Ferrantelli Flavia, Manfredi Francesco, Chiozzini Chiara, Leone Patrizia, Giovannelli Andrea, Olivetta Eleonora, Federico Maurizio
National Center for Global Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
National Center for Animal Experimentation and Welfare, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Cancers (Basel). 2021 May 8;13(9):2263. doi: 10.3390/cancers13092263.
We developed an innovative method to induce antigen-specific CD8 T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This approach employs a DNA vector expressing a mutated HIV-1 Nef protein (Nef) deprived of the anti-cellular effects typical of the wild-type isoform, meanwhile showing an unusual efficiency of incorporation into EVs. This function persists even when foreign antigens are fused to its C-terminus. In this way, Nef traffics large amounts of antigens fused to it into EVs spontaneously released by the recipient cells. We previously provided evidence that mice injected with a DNA vector expressing the Nef/HPV16-E7 fusion protein developed an E7-specific CTL immune response as detected 2 weeks after the second immunization. Here, we extended and optimized the anti-HPV16 CD8 T cell immune response induced by the endogenously engineered EVs, and evaluated the therapeutic antitumor efficacy over time. We found that the co-injection of DNA vectors expressing Nef fused with E6 and E7 generated a stronger anti-HPV16 immune response compared to that observed in mice injected with the single vectors. When HPV16-E6 and -E7 co-expressing tumor cells were implanted before immunization, all mice survived at day 44, whereas no mice injected with either void or Nef-expressing vectors survived until day 32 after tumor implantation. A substantial part of immunized mice (7 out of 12) cleared the tumor. When the cured mice were re-challenged with a second tumor cell implantation, none of them developed tumors. Both E6- and E7-specific CD8 T immunities were still detectable at the end of the observation time. We concluded that the immunity elicited by engineered EVs, besides counteracting and curing already developed tumors, was strong enough to guarantee the resistance to additional tumor attacks. These results can be of relevance for the therapy of both metastatic and relapsing tumors.
我们开发了一种基于细胞外囊泡（EVs）体内工程改造来诱导抗原特异性CD8 T细胞毒性淋巴细胞（CTL）免疫的创新方法。该方法采用一种表达突变型HIV-1 Nef蛋白（Nef）的DNA载体，该蛋白缺乏野生型异构体典型的抗细胞效应，同时显示出异常高效地掺入EVs。即使将外源抗原融合到其C末端，这种功能仍然存在。通过这种方式，Nef将大量与其融合的抗原运输到受体细胞自发释放的EVs中。我们之前提供的证据表明，注射表达Nef/HPV16-E7融合蛋白的DNA载体的小鼠在第二次免疫后2周检测到产生了E7特异性CTL免疫反应。在这里，我们扩展并优化了内源性工程改造的EVs诱导的抗HPV16 CD8 T细胞免疫反应，并随时间评估了治疗性抗肿瘤疗效。我们发现，与注射单一载体的小鼠相比，共注射表达与E6和E7融合的Nef的DNA载体产生了更强的抗HPV16免疫反应。在免疫前植入共表达HPV16-E6和-E7的肿瘤细胞时，所有小鼠在第44天存活，而注射空载体或表达Nef载体的小鼠在肿瘤植入后直到第32天均无存活。相当一部分免疫小鼠（12只中的7只）清除了肿瘤。当治愈的小鼠再次接受第二次肿瘤细胞植入挑战时，它们均未发生肿瘤。在观察期结束时，仍可检测到E6和E7特异性CD8 T免疫。我们得出结论，工程改造的EVs引发的免疫除了能对抗和治愈已形成的肿瘤外，还强大到足以保证对额外肿瘤攻击的抗性。这些结果对于转移性和复发性肿瘤的治疗可能具有重要意义。
