靶向PD-1/PD-L1通路的溶瘤痘苗病毒——抗体治疗策略对B细胞淋巴瘤的杀伤作用研究

Programmed death 1 (PD-1) is an inhibitory receptor expressed on the surface of T cells, which physiologically limits T-cell activation and proliferation. A important finding that encouraged the development of monoclonal antibodies blocking the PD-1 pathway for treating relapsed/refractory B cell lymphoma was that PD-1 ligands (PL-L1 and PD-L2) are overexpressed in B cell lymphoma, whereas PD-1 is upregulated on tumor-infiltrating lymphocytes. However, previous clinic trials showed that majority of patients treated with anti-PD-1/PD-L1 monotherapies do not achieve objective responses. Furthermore, another challenging problems with the use of monoclonal antibodies specific for PD-1 is the management of severe autoimmune side effects. Recent studies suggest that combinations of anti-PD-1/PD-L1 agents with other antitumor agents such as chemotherapeutic agents, small molecular inhibitors and oncolytic viruses improve anti-tumor responses, which may represent a right path to achieve cancer cure.. Oncolytic vaccinia viruses (OVVs) are promising anticancer agents owing to their ability to infect, replicate in, and lyse tumor cells and spread to other cancer cells without causing significant adverse events in the cancer patients. In addition, OVVs have been used as a gene therapy platform to deliver anti-cancer genes, to enhance the anticancer effects of the OVV. Previously, we combined the miR-34a that induces apoptosis with OVV, and demonstrated in vitro and in vivo antileukemia activity of the virus can be significantly improved. Furthermore, we construct a vector coding for a fully human monoclonal antibody specific for PD-1 using a linker, that is, sequence encoding the four amino acids IETD, to join heavy and light chain of PD-1 antibody genes, and demonstrated that the supernatants collected from the culture of 293A cells transduced with the vector contain proteins with anti-PD-1 antibody activity, thereby providing a useful tool to construct an OVV carrying anti-PD-1 antibody gene. In this study, we will established a new OVV that is armed with the gene encoding a fully human monoclonal antibody specific for PD-1, and study infectivity and anti-tumor activity of this OVV on B cell lymphoma via blocking PD-1 pathway and oncolytic effect of the virus. Next, we will construct another novel OVV that express both human anti-PD-1 monoclonal antibody and p53 genes. This design is based on the data showing that PD-L1 expression is regulated by p53/miR-34 axis. The insertion of anti-PD-1 antibody and p53 genes into the genome of a modified OVV shares the advantages of gene therapy, dual-targeting PD-1/PD-L1 axis, and virotherapy, which can not only directly kill cancer cells by oncolysis, but also completely block PD-1/PD-L1 pathway, resulting in a potent activity against lymphoma.

靶向免疫检查点PD-1/PD-L1是治疗难治性B淋巴瘤的重要新方法。联合疗法是克服PD-1单抗疗法局限性即多数病人不能获得持久反应、严重的免疫相关副作用等的重要手段。新近研究表明，免疫检查点抑制疗法和溶瘤病毒联合可能是有前景的策略。溶瘤痘苗病毒（OVV）既有溶瘤又可携带治疗性基因增强其抗癌效应，具有全身抗肿瘤、破坏肿瘤床等优点。前期我们构建了携带外源基因的新型OVV，证明其有很好的抗血液肿瘤作用。为了实现溶瘤病毒-抗体联合策略，我们应用IETD连接子构建了表达人PD-1抗体的穿梭质粒，并证明该质粒感染293细胞后的上清中含有PD-1抗体活性。本项目将OVV携带人PD-1单抗基因；应用人源化淋巴瘤小鼠模型研究抑制PD-1的OVV-抗体治疗策略在治疗B淋巴瘤中的作用；通过同源重组使OVV同时表达人PD-1单抗基因和p53，后者通过miR-34抑制PD-L1表达；实现双靶向PD-1/PD-L。

阻断免疫检查点PD-1/PD-L1是治疗难治性B淋巴瘤的重要新方法。然而严重的免疫相关副作用以及多数患者的疾病复发限制了PD-1/PD-L1单抗和抑制剂的临床应用。溶瘤痘苗病毒（OVV）既有溶瘤又可携带治疗性基因增强其抗癌效应，又具有全身抗肿瘤、破坏肿瘤床等优点。因此本课题提出联合OVV和PD-1/PD-L1阻滞的新策略，通过该项目的实施我们成功构建了3种阻断PD1/PD-L1的OVV：携带人PD-1全长抗体序列的OVV-anti-PD-1fl、携带人PD-1抗体scFv序列的OVV-anti-PD-1 scFv和携带人PD-L1抗体scFv序列的OVV-anti-PD-L1 scFv；分别通过流式细胞术、western blot和ELISA等实验方法证明了该3种抗PD1/PD-L1的OVV表达相应抗体和阻断PD1/PD-L1信号通路的能力；通过检测细胞存活、细胞因子的分泌以及构建小鼠模型等证明了上述3种抗PD1/PD-L1的OVV在细胞水平和小鼠水平抗B细胞淋巴瘤细胞和激发机体免疫应答的能力。综上所述，本项目基本完成了项目计划书的研究内容，并达到相应的研究目标，实现溶瘤病毒和PD-1/PD-L1抗体的联合治疗。