细胞载体介导溶瘤腺病毒瘤内活化记忆性自然杀伤细胞细胞靶向治疗实体瘤的研究

The recurrence and metastasis of tumors are one of the most thorny problems in the clinical settings and approaches to treatment seem to be extremely limited. Cancer immunotherapy would provide a new hope with solving such a clinical problem. Antitumor activities of oncolytic adenovirus (OA) mediated by cells and cell-derived microparticles as carriers had been investigated in our previous two NSFCs, and the enhanced antitumor activity of OA was observed via mechanisms by which decrease in the immunogenicity of OA and increase in the tumor targeting of OA. However, owing to OA itself and complex biological properties of tumors, only relying on OA to eliminate tumors are obviously impossible. In this study, with the utilize of OA itself being a potentially excellent vector, a serial of OA vectors encoding inmunoregulatory factors will be constructed for selectively expanding and activating memory natural killer in vivo. The ability to genetically engineered cell carrier-mediated targeting delivery of OA constructs by systemic administration to tumor beds, including distant metastatic tumor sites, will be validated in the model of tumor-bearing mice, and effect of the OAs on the expansion and activation of memory NK cells in vivo and synergetic antitumor activity of the OAs with memory NK cells will be investigated in the tumor immunosuppressive microenvironment. In addition, further investigation in the phenotypic characteristics and functions of tumor in situ activation of memory NK cells and antitumor mechanisms will be made, providing the theoretical and experimental basis for this new targeting strategy to solid tumors.

肿瘤的复发或转移是临床所面临最棘手问题之一，治疗手段十分有限，免疫治疗为解决这一临床难题提供了新希望。我们之前的两个国基金探讨了以细胞及细胞来源的微颗粒作载体介导溶瘤腺病毒（OA）的抗肿瘤效应，证实微颗粒和细胞能通过降低OA的免疫原性、增强靶向性等机制显著增强其抗肿瘤活性。但由于OA自身及肿瘤复杂的生物学特性，单纯利用OA达到消除肿瘤的作用显然有限。本研究拟结合OA本身即是一种优质载体的特性，构建系列编码免疫调节因子从而能选择性扩增、激活记忆性NK细胞的OA载体，在荷瘤小鼠模型中，利用基因工程细胞载体，将这些OA通过系统给药方式靶向转送至肿瘤，包括远处转移的肿瘤部位，观察这些OA在肿瘤免疫抑制微环境中扩增、活化记忆性NK细胞的作用以及OA联合记忆性NK细胞的抗肿瘤效果，并对肿瘤原位活化记忆性NK细胞的表型和功能特征、抗肿瘤活性机制进行探讨，为这一实体瘤靶向治疗新策略提供理论基础和实验依据。

细胞治疗在肿瘤治疗领域取得突破性进展，其中，病毒-细胞治疗是肿瘤研究的重要方向。NK细胞作为机体的第一道防线，在肿瘤作用中具有显著优势，然而其在肿瘤组织中的浸润数量有限且处于功能失活状态。溶瘤腺病毒本身即为一种优质载体,具有溶瘤效果好、安全性高和制备方便等优点。本研究采用溶瘤腺病毒和NK细胞相结合的策略。一方面利用溶瘤腺病毒的溶瘤效应；另一方面利用溶瘤腺病毒载体编码系列免疫调控因子来选择性扩增和激活肿瘤内的NK细胞，增强二者的协同抗肿瘤作用。首先通过CRISPR-Cas9基因编辑技术对白血病K562细胞进行多基因编辑和修饰，建立了表达IL-2、4-1BBL和膜型IL-21的K562细胞（K562-IL-2/4-1BBL/mIL-21），用于体外NK细胞的扩增和激活。构建人端粒酶启动子（hTERTp）驱动E1A和细胞因子的溶瘤腺病毒Ad5 TERTp-E1A-CMVp-IL-2/4-1BBL/mIL-21载体，通过体内外实验，检测溶瘤腺病毒的抗肿瘤效果及对NK细胞的影响。结果显示，K562-IL-2/4-1BBL/mIL-21细胞体外能够有效扩增外周血单核细胞来源的NK细胞，并且扩增的NK细胞被激活且具有抗肿瘤活性；溶瘤腺病毒Ad5 TERTp-E1A-CMVp-IL-2/4-1BBL/mIL-21具有显著抗肿瘤效果并增加NK细胞在肿瘤组织中的浸润。该研究为增强抗肿瘤免疫、靶向肿瘤治疗提供新的策略和理论依据，具有重要的研究和临床意义。