腺苷A2受体拮抗剂协同蜂毒肽溶瘤作用的靶向纳米共递送系统构建和机制研究

Anoplin (ANO), an oncolytic peptide isolated from spider wasp Anoplius samariensis, could effectively disintegrate tumor cells in cell circle-independent manner with release of cancer specific antigens accompanied by activation of anti-tumor immune response. However, the simultaneously released ATP and its metabolite adenosine could induce the serious tumor immunosuppressive microenvironment (TIM). To date, the approach to reverse the TIM induced by oncolytic peptide treatment has not been documented yet. We previously confirmed that the adenosine A2 receptor (A2AR) antagonist CPI-444 (CPI) could effectively improve the TIM in the ANO treated triple-negative breast cancer (TNBC). Hence, we proposed that the A2AR could serve as the novel target for synergistic treatment with ANO in TNBC. In this study, the co-delivery TNBC-targeted nanomedicines of ANO and CPI are developed to produce tumor cell oncolysis with improved TIM, and to realize the synchronically synergistic effects. For the first time, the reprograming effect of CPI on TIM induced by ANO and the underlying mechanism will be elucidated by the in vitro and in vivo study. Our investigation will provide experimental evidence for the Trimodal therapeutic strategy with targeted delivery, efficient oncolysis and TIM reprograming.

蜂毒肽（Anoplin，ANO）是一种细胞周期非依赖性的溶瘤肽，其裂解肿瘤细胞后释放的特异性抗原可激活抗肿瘤免疫。然而，伴随细胞裂解所释放ATP的降解产物腺苷亦会诱导肿瘤免疫抑制性微环境（TIM）的产生，如何重塑溶瘤肽引起的TIM尚未见报道。前期预实验表明，腺苷A2受体拮抗剂CPI可明显逆转ANO诱导三阴性乳腺癌（TNBC）产生的TIM。为此，我们探索ANO和CPI肿瘤靶向纳米共递送系统，以期实现靶向溶瘤和同步改善TIM的协同效应。本研究中，构建和表征以iRGD多肽为靶向配体的ANO-CPI共递送纳米系统；开展对TNBC细胞4T1的体外溶瘤作用和免疫细胞调控作用的研究；建立荷瘤模型研究体内靶向溶瘤和重塑TIM的协同效应和机制。研究成果将揭示腺苷A2受体拮抗剂作为新型免疫微环境调节剂重塑溶瘤肽诱导的TIM效应的机制，并为靶向递送、高效溶瘤和TIM改善“三位一体”肿瘤治疗新策略提供实验依据。

蜂毒肽（Anoplin，ANO）是一种细胞周期非依赖性的溶瘤肽，其裂解肿瘤细胞后释放的特异性抗原可激活抗肿瘤免疫。然而，肿瘤细胞裂解所释放的ATP代谢产物腺苷亦会诱导肿瘤免疫抑制性微环境（TIM）的产生，如何重塑溶瘤肽引起的TIM具有重要意义。前期预实验表明，腺苷A2受体拮抗剂CPI可明显逆转蜂毒溶瘤肽诱导三阴性乳腺癌（TNBC）产生的TIM。为此，我们将腺苷A2受体拮抗剂CPI作为新型TIM调节剂，成功构建了TNBC细胞靶向的蜂毒溶瘤肽和CPI共递送纳米系统CA@TLM。随后通过体内外实验，系统评价了CA@TLM的肿瘤靶向性、溶瘤特性、免疫原性细胞死亡（ICD）诱导作用及对免疫细胞的调控。体外实验结果显示：CA@TLM可被TNBC细胞主动摄取，并成功裂解细胞膜，引发ICD；同时，克服腺苷造成的免疫抑制，保护M1型巨噬细胞，逆转M2型巨噬细胞的极化，促进DC细胞成熟。体内动物实验表明CA@TLM可有效靶向TNBC肿瘤，抑制肿瘤生长，促进CD8+ T细胞的肿瘤浸润，增强溶瘤免疫治疗效果。本研究揭示了CPI作为免疫微环境调节剂改善TIM效应的机制，为有效实现靶向递送、高效溶瘤和TIM改善“三位一体”肿瘤治疗新策略提供了实验依据。在本项目的资助下发表高水平SCI论文（IF 5分以上）5篇，申请国家发明专利2项，培养研究生4名。

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