抗PD-1抗体SCFV基因重组水泡口炎病毒（VSV-Anti-PD-1）抗肿瘤作用及免疫机制研究

Immune costimulatory molecule PD-1， as a negative regulator of immune，plays a key role in mediating tumor immune escape with high expression in immune cells in the tumor microenvironment resulting in an inhibitory immune microenvironment. Clinical studies have demonstrated that targeting PD-1 signal can break immune tolerance and bring clinical benefit in cancer treatment. However, systemic administration inducing immune disease and expensive costs limit its application. Therefore, local anti- PD-1 treatment is expected to be better immune therapy with less toxicity and cost compared of systemic administration. Using oncolytic viruses to local express active anti-PD-1 SCFV fragment is a valid choice. We previously have demonstrated vesicular stomatitis virus (VSV) therapy can effectively promote tumor cell apoptosis and affect tumor immune microenvironment. With successful importing epidermal growth factor, VSV is the ideal therapy vectors. Intratumoral injection of VSV-Anti-PD-1 recombinant virus can express the Anti-PD-1 active fragments and regulate the immune microenvironment by inhibiting PD-1 signal and activating the anti- tumor immunity. Implementation of this project is expected to verify the anti-tumor efficacy of VSV-Anti-PD-1 recombinant virus and analyze related immune mechanisms, which provides a new security, effective treatment strategies for tumor immunotherapy of Anti-PD-1.

免疫共刺激分子PD-1作为负性调节分子，在肿瘤微环境的免疫细胞中高表达，能形成抑制性免疫微环境，在肿瘤免疫逃逸中起关键作用。已证实抗PD-1能够打破免疫耐受，给患者带来获益，但全身给药诱发的免疫相关性疾病以及高额的费用限制了其应用。故肿瘤微环境局部抗PD-1治疗有望更好激活免疫，且减少全身给药毒性并降低花费，采用溶瘤病毒局部表达抗PD-1抗体SCFV活性片段是一个有效方法。我们前期证实水泡口炎病毒（VSV）能有效促进肿瘤细胞凋亡和影响肿瘤免疫微环境，且成功导入生长因子等基因，是一种理想的基因导入载体。本课题拟通过瘤内导入抗PD-1抗体SCFV活性片段和VSV的重组病毒（VSV-Anti-PD-1），达到局部高表达抗PD-1活性片段来抑制PD-1信号、激活抗肿瘤免疫。本项目将探索VSV-Anti-PD-1重组病毒的靶向投递效果及抗肿瘤作用机制，为抗PD-1免疫治疗提供全新的安全、有效的治疗策略。

溶瘤病毒治疗有效性受限于肿瘤免疫抑制性微环境，免疫检查点（PD-1/PD-L1）抑制剂联合溶瘤病毒治疗有望进一步激活免疫反应、改善抗肿瘤疗效。本研究使用HSV-2作为载体，构建了表达人源化PD-1抗体序列的新型溶瘤病毒（oHSV2-ahPD1），并在临床前三阴性乳腺癌模型中评估了其抗肿瘤作用及安全性。在体外实验中，oHSV2和oHSV2-ahPD1均能感染并溶解了三阴性乳腺癌细胞系。在Balb/c-hPdcd1小鼠模型中，瘤内注射oHSV2-ahPD1较oHSV2或PBS明显减缓4T-1肿瘤的生长。进一步探索潜在的机制，发现oHSV2-ahPD1可能通过激活CD3+CD4+，CD3+CD8+T细胞增殖以及降低CD11b+Gr-1+MDSC浸润以改善疗效。肿瘤组织RNA-seq提示关键参与的KEGG通路包括细胞因子-细胞因子受体相互作用和趋化因子信号传导等。在生物安全性方面，oHSV2或oHSV2-ahPD1治疗未导致明显异常的镜下组织病理学或血液生化学改变。综上所述，oHSV2-ahPD1可能成为三阴性乳腺癌的有效、安全的治疗策略。