新型肺癌抗原LunX免疫原性的体内增强机制与抗体协同治疗研究

Inducing the immunogenicity of tumor cells is an important means to initiate the anti-tumor immune response. It is rarely reported that a novel tumor specific immunogen was found and induced to display stronger immunogenicity in vivo. The applicant first reported that lung specific X protein (LunX) was closely related to the malignancy of lung cancer. And in humanized mice, it has been proved that the monoclonal antibody targeting LunX can inhibit the growth of human lung cancer (Zheng... Cancer Res 2015). Our recent results show that, calreticulin (CRT) can promote the immunogenicity of LunX by turning it out to the cell membrane. The aim of this study was to establish a double human lung cancer model (PDC/PDX) and K-RasG12D spontaneous lung cancer model in mice. To further study the mechanism of low dose small molecular drugs to enhance the immunogenicity of lung cancer cells, and to explore the mechanism of CRT adjuvant immunogen LunX presented on the membrane surface, and then to observe the role of dendritic cells (DC) in the anti-tumor immunity. In order to expand the low-dose or single-dose chemotherapy combined with LunX antibody drugs for lung cancer treatment of new schemes and new strategies.

诱导肿瘤细胞的免疫原性是启动抗肿瘤免疫应答的重要手段，其中发现新型肿瘤特异性免疫原并体内诱导其展示更强免疫原性则鲜有报道。申请人曾率先报道肺特异X蛋白(LunX)与肺癌恶性程度密切相关，并在人源化小鼠证明靶向LunX的单抗可以抑制人肺癌生长(Zheng…Cancer Res 2015)。我们最近的结果显示，钙网蛋白（CRT）可协助LunX外翻至细胞膜以促进其免疫原性。本研究拟在双人源化肺癌小鼠模型（PDC+PDX）和K-RasG12D自发肺癌小鼠模型，进一步研究低剂量小分子药物增强肺癌细胞免疫原性的机制，深入探讨内质网腔内的CRT辅助免疫原LunX提呈于膜表面的机制，并进而观察树突状细胞（DC）在该抗肿瘤免疫中的作用，为拓展低剂量或单次化疗联合LunX抗体药物治疗肺癌的新方案与新策略。

为了探究LUNX蛋白在肺癌免疫原性化疗过程中的变化，我们利用不同剂量的免疫化疗药物米托蒽醌对非小细胞肺癌细胞系进行处理，并通过免疫荧光检测细胞膜表面的LUNX蛋白表达变化。我们发现，低剂量的米托蒽醌处理可以显著上调细胞膜表面的LUNX蛋白表达，但是不影响细胞整体的LUNX表达；并且LUNX蛋白表达上调的肿瘤细胞出现了细胞体积增大，细胞内颗粒复杂程度增加的典型非凋亡形态变化，证明免疫化疗诱导LUNX蛋白向细胞膜转移的过程不依赖于细胞凋亡。.我们在体内验证了anti-LUNX清除衰老肿瘤细胞的能力。我们首先发现，免疫化疗同样可以在体内诱导肿瘤细胞膜表面LUNX蛋白表达增加，并且免疫化疗联合anti-LUNX可以有效抑制肺癌细胞皮下瘤模型的生长；更进一步，我们将衰老的肿瘤细胞直接植入小鼠体内，并在小鼠成瘤后对小鼠进行anti-LUNX治疗。我们发现，anti-LUNX可以有效抑制衰老肿瘤细胞皮下瘤和转移瘤模型的生长；为了验证抗体依赖的自然杀伤细胞介导的细胞毒效应对衰老细胞的清除效果，我们在上述治疗体系中加入自然杀伤细胞，发现与单独使用anti-LUNX相比，联合自然杀伤细胞和anti-LUNX可以更有效地清除小鼠体内衰老的肿瘤细胞。.综上所述，我们证明了免疫化疗可以诱导肺癌细胞发生细胞衰老，并促进LUNX蛋白和自然杀伤细胞活化性受体对应的配体向细胞膜转移；通过anti-LUNX靶向发生免疫原性衰老的肿瘤细胞，一方面可以通过anti-LUNX的直接作用抑制衰老肿瘤细胞生存；另一方面还可以通过anti-LUNX依赖的自然杀伤细胞介导的细胞毒作用清除衰老的肿瘤细胞。.我们的研究为肺癌临床免疫治疗提供了新的思路：即先通过免疫化疗诱导肿瘤细胞发生免疫原性衰老，增强肺癌的免疫原性；再利用肿瘤特异性单克隆抗体或者其他免疫疗法杀伤衰老的肿瘤细胞。