PDF(Pubmed)
Abstract：
Toxoplasma gondii holds significant therapeutic potential; however, its nonspecific invasiveness results in off-target effects. The purpose of this study is to evaluate whether T. gondii specificity can be improved by surface display of scFv directed against dendritic cells\' endocytic receptor, DEC205, and immune checkpoint PD-L1. Anti-DEC205 scFv was anchored to the T. gondii surface either directly via glycosylphosphatidylinositol (GPI) or by fusion with the SAG1 protein. Both constructs were successfully expressed, but the binding results suggested that the anti-DEC-SAG1 scFv had more reliable functionality towards recombinant DEC protein and DEC205-expressing MutuDC cells. Two anti-PD-L1 scFv constructs were developed that differed in the localization of the HA tag. Both constructs were adequately expressed, but the localization of the HA tag determined the functionality by binding to PD-L1 protein. Co-incubation of T. gondii displaying anti-PD-L1 scFv with tumor cells expressing/displaying different levels of PD-L1 showed strong binding depending on the level of available biomarker. Neutralization assays confirmed that binding was due to the specific interaction between anti-PD-L1 scFv and its ligand. A mixed-cell assay showed that T. gondii expressing anti-PD-L1 scFv predominately targets the PD-L1-positive cells, with negligible off-target binding. The recombinant RH-PD-L1-C strain showed increased killing ability on PD-L1+ tumor cell lines compared to the parental strain. Moreover, a co-culture assay of target tumor cells and effector CD8+ T cells showed that our model could inhibit PD1/PD-L1 interaction and potentiate T-cell immune response. These findings highlight surface display of antibody fragments as a promising strategy of targeting replicative T. gondii strains while minimizing nonspecific binding.
摘要：
弓形虫具有重要的治疗潜力；然而，其非特异性侵袭性导致脱靶效应。这项研究的目的是评估弓形虫特异性是否可以通过表面展示针对树突状细胞的scFv来提高，DEC205和免疫检查点PD-L1。抗DEC205scFv直接经由糖基磷脂酰肌醇(GPI)或通过与SAG1蛋白融合而锚定至弓形虫表面。两个构建体都成功表达，但是结合结果表明，抗DEC-SAG1scFv对重组DEC蛋白和表达DEC205的MutuDC细胞具有更可靠的功能。开发了两种在HA标签的定位上不同的抗PD-L1scFv构建体。两种结构都得到了充分表达，但是HA标签的定位决定了与PD-L1蛋白结合的功能。显示抗PD-L1scFv的弓形虫与表达/显示不同水平的PD-L1的肿瘤细胞的共孵育显示强结合,这取决于可用生物标志物的水平。中和测定证实,结合是由于抗PD-L1scFv与其配体之间的特异性相互作用。混合细胞试验表明，表达抗PD-L1scFv的弓形虫主要靶向PD-L1阳性细胞，具有可忽略的脱靶结合。与亲本菌株相比，重组RH-PD-L1-C菌株对PD-L1肿瘤细胞系的杀伤能力增加。此外，靶肿瘤细胞和效应CD8+T细胞共培养试验表明,我们的模型可以抑制PD1/PD-L1相互作用并增强T细胞免疫应答.这些发现强调了抗体片段的表面展示作为靶向复制性弓形虫菌株同时最小化非特异性结合的有希望的策略。
