放射疗法(RT)引发免疫原性细胞死亡(ICD)。L-ASNase,催化天冬酰胺(Asn)的转化,从而耗尽它,用于治疗血癌。在以前的工作中,我们发现CRT3LP和CRT4LP,与钙网蛋白(CRT)特异性单体CRT3和CRT4缀合的PAS化L-ASN酶增加了ICD诱导化疗的功效。这里,我们评估了它们在接受RT治疗的荷瘤小鼠中的疗效。方法:通过计算机分子对接分析评估单体结合。通过共聚焦成像和流式细胞术评估ecto-CRT的表达和细胞定位。通过ELISA分析CRT3LP和CRT4LP在辐射(IR)诱导的肿瘤ICD中的抗肿瘤作用和作用。免疫组织化学,免疫分析方法。结果:分子对接分析表明CRT3和CRT4单体与CRT稳定结合。暴露于10GyIR以时间依赖性方式降低CT-26和MC-38肿瘤细胞的活力,直至72h,并增加ICD标记物ecto-CRT(暴露在细胞表面的CRT)和免疫检查点标记物PD-L1的表达,直到48小时。IR增强了CT-26和MC-38肿瘤细胞中CRT3LP和CRT4LP的细胞毒性,和增加活性氧(ROS)水平。在用6GyIR治疗的携带CT-26和MC-38皮下肿瘤的小鼠中,Rluc8缀合的CRT特异性单体(CRT3-Rluc8和CRT4-Rluc8)特异性靶向肿瘤组织,CRT3LP和CRT4LP增加了肿瘤组织中的总ROS水平,从而增强RT的抗肿瘤功效。这些小鼠的肿瘤组织显示成熟树突状细胞增多,CD4+T,和CD8+T细胞和促炎细胞因子(IFNγ和TNFα)和减少的调节性T细胞,肿瘤细胞增殖标志物(Ki67和CD31)表达下调。这些数据表明IR和CRT靶向L-ASN酶的组合激活并重新编程肿瘤微环境的免疫系统。与这些数据一致,免疫检查点抑制剂(抗PD-L1抗体)显著提高了联合IR和CRT靶向L-ASNase的疗效.结论:CRT特异性L-ASNases可用作RT治疗肿瘤的候选药物。与抗PD-L1抗体的联合治疗增加了它们的治疗功效。
Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4+ T, and CD8+ T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.