我们之前开发了一种创新策略，通过细胞外囊泡 (EV) 的体内工程诱导 CD8 T 淋巴细胞免疫。这种方法依赖于肌内注射表达感兴趣抗原的 DNA，该抗原融合在无生物活性的 HIV-1 Nef 蛋白突变体 (Nefmut) 上。 Nefmut 非常有效地整合到 EV 中，从而将大量融合蛋白输送到转染细胞释放的 EV 中。事实证明，该平台能够成功对抗高免疫原性肿瘤特异性抗原。在这里，我们测试了工程化 EV 诱导的抗原特异性 CD8 T 细胞免疫反应是否可以抵消表达两种“自身”肿瘤相关抗原 (TAA)：HOXB7 和 Her2/neu 的肿瘤的生长。在皮下植入共表达 HOXB7 和 Her2/neu 的乳腺癌细胞之前，给 FVB/N 小鼠单独或组合注射表达与 HOXB7 或 Her2/neu 融合的 Nefmut 的 DNA 载体。所有接种联合疫苗的小鼠均保持无肿瘤状态，而接种单一 Nefmut 融合抗原的小鼠仅得到部分保护，而 Her2/neu 免疫小鼠的抗肿瘤作用更强。双重疫苗接种的小鼠在随后的肿瘤细胞再次攻击后也控制了肿瘤生长。重要的是，联合疫苗接种在治疗性免疫环境中也包含肿瘤。这些结果显示了基于 EV 的疫苗接种针对两种 TAA 的功效，并首次证明 HOXB7 可能是多抗原免疫治疗策略的目标。© 2024。作者。

We previously developed an innovative strategy to induce CD8+ T lymphocyte-immunity through in vivo engineering of extracellular vesicles (EVs). This approach relies on intramuscular injection of DNA expressing antigens of interest fused at a biologically-inactive HIV-1 Nef protein mutant (Nefmut). Nefmut is very efficiently incorporated into EVs, thus conveying large amounts of fusion proteins into EVs released by transfected cells. This platform proved successful against highly immunogenic tumor-specific antigens. Here, we tested whether antigen-specific CD8+ T cell immune responses induced by engineered EVs can counteract the growth of tumors expressing two "self" tumor-associated antigens (TAAs): HOXB7 and Her2/neu. FVB/N mice were injected with DNA vectors expressing Nefmut fused to HOXB7 or Her2/neu, singly and in combination, before subcutaneous implantation of breast carcinoma cells co-expressing HOXB7 and Her2/neu. All mice immunized with the combination vaccine remained tumor-free, whereas groups vaccinated with single Nefmut-fused antigens were only partly protected, with stronger antitumor effects in Her2/neu-immunized mice. Double-vaccinated mice also controlled tumor growth upon a later tumor cell re-challenge. Importantly, co-vaccination also contained tumors in a therapeutic immunization setting. These results showed the efficacy of EV-based vaccination against two TAAs, and represent the first demonstration that HOXB7 may be targeted in multi-antigen immunotherapy strategies.© 2024. The Author(s).