靶向纳米级免疫偶联物诱导检查点抑制剂抗体治疗颅内胶质瘤的可行性实验研究

目的 探讨靶向纳米级免疫偶联物(Nanoscale immunoconjugates,NCI)诱导检查点抑制剂抗体治疗颅内胶质瘤的可行性。方法 无特定病原体级(Specific pathogen free,SPF)级、雌性大鼠168只,随机分为磷酸缓冲盐溶液(Phosphate buffer saline,PBS)组(24只),游离组(72只)及NIC组(72只); 所有大鼠均进行颅内胶质瘤细胞植入,构建颅内胶质瘤大鼠模型; PBS组不进行治疗; 游离组给予T淋巴细胞相关抗原4(Cytotoxic T lymphocyte associated antigen-4,a-CTLA-4)和程序性细胞死亡1(Anti programmed cell death protein-1,a-PD-1)及两者联合注射,每种各24只; NCI组给予纳米级免疫偶联物诱导检查点抑制剂抗体[分别为聚苹果酸/ T淋巴细胞相关抗原4(Polymalic malic acid/ Cytotoxic T lymphocyte associated antigen-4,P/a-CTLA-4)、聚苹果酸/程序性细胞死亡1(Polymalic malic acid/Anti programmed cell death protein-1,P/a-PD-1)及两盒联合注射]注射,每种各24只; 使用荧光素标记法观察不同组大鼠药物血脑屏障穿透效率,比较不同治疗方式大鼠治疗后CD3+T细胞(CD3+Pan T Cells,CD3+)、CD4+,CD8+、调节性T细胞(Regulatory T cells,Treg)、巨噬细胞(MΦ)、自然杀伤细胞(Natural killer cell,NK)细胞、自然杀伤T细胞(Natural killer T cell,NKT)细胞、干扰素γ(Interferon-γ,IFNγ)水平及大鼠CD4+,CD8+增殖活跃程度、生存期。结果 荧光实验显示,NIC组各治疗方式大鼠脑部荧光面积均显著高于游离组及PBS组; NCI各组CD3+、CD4+、CD8+、Treg、CD4+ki67、CD8+ki67、MΦ、M1MΦ、M2MΦ、NK细胞、NKT细胞、IFNγ每孔计数及总体生存期显著高于游离组及PBS组(P<0.05)。结论 NCI诱导检查点抑制剂抗体能促进药物透过血脑屏障,刺激大脑驻留的免疫系统,促使CD8+ T细胞增殖并触发多种免疫细胞因子的释放,增加M1型巨噬细胞的产生,从而协调针对GBM的免疫反应,提高颅内胶质瘤大鼠存活时间。

An study on the treatment of intracranial glioma with checkpoint inhibitor antibodies induced by targeting nanoscale immunoconjugates

Objective To explore the feasibility of treatment of intracranial gliomas with checkpoint inhibitor antibodies induced by targeting nano-scale immunoconjugates(NCI).Methods 252 SPF female rats were randomly divided into PBS group(24 rats), free group(72 rats)and NIC group(72 rats). All rats were implanted with intracranial glioma cells to construct Intracranial glioma rat model, the PBS group was treated with PBS, and the free group was given T lymphocyte-associated antigen 4(a-CTLA-4)or programmed cell death 1(a-PD-1)injection separately and in combination. each group contains 24 rats, the NCI group was given nano-scale immunoconjugate-induced checkpoint inhibitor antibodies(polymalic malic acid/ cytotoxic T lymphocyte associated antigen-4(P/a-CTLA-4)and polymalic malic acid/Anti Programmed cell death protein-1(P/a-PD-1), separately and in combination))injections, each group contains 24 rats. The blood-brain barrier penetration efficiency was observed by fluorescein-labeling method. The CD3+, CD4+, CD8+ T cells, Treg, macrophages(MΦ), NK cells, NKT cells after treatment in rats with different treatment methods, IFNγ level and rat CD4+, CD8+ proliferation activity and survival time was compared in the different groups.Results Fluorescence experiment showed that the brain fluorescence area of rats in the NIC group was significantly wider than that in the free group and the PBS group. The number of CD3+ T cells, CD4+ T cells, CD8+ T cells, Treg, CD4+ ki67 cells, CD8+ ki67 cells, MΦ, M1MΦ, M2MΦ, NK cells and NKT cells, IFNγ counts per well and overall survival period in NCI group were significantly higher than those in the free and PBS group(P<0.05).Conclusion NCI-induced checkpoint inhibitor antibodies can promote drugs to penetrate the blood-brain barrier, stimulate the immune system resident in the brain, promote the proliferation of CD8 + T cells, trigger the release of various immune cytokines, and increase the production of M1 macrophages, in order to coordinate the immune response against GBM and improve the survival time of intracranial glioma rats.