regorafenib 通过抑制肿瘤相关巨噬细胞中的 p38 激酶/Creb1/Klf4 轴增强抗肿瘤免疫。

BACKGROUND: Regorafenib and other multikinase inhibitors may enhance antitumor efficacy of anti-program cell death-1 (anti-PD1) therapy in hepatocellular carcinoma (HCC). Its immunomodulatory effects, besides anti-angiogenesis, were not clearly defined. METHODS: In vivo antitumor efficacy was tested in multiple syngeneic liver cancer models. Murine bone marrow-derived macrophages (BMDMs) were tested in vitro for modulation of polarization by regorafenib and activation of cocultured T cells. Markers of M1/M2 polarization were measured by quantitative reverse transcription PCR (RT-PCR), arginase activity, flow cytometry, and ELISA. Knockdown of p38 kinase and downstream Creb1/Klf4 signaling on macrophage polarization were confirmed by using knockdown of the upstream MAPK14 kinase, chemical p38 kinase inhibitor, and chromatin immunoprecipitation. RESULTS: Regorafenib (5 mg/kg/day, corresponding to about half of human clinical dosage) inhibited tumor growth and angiogenesis in vivo similarly to DC-101 (anti-VEGFR2 antibody) but produced higher T cell activation and M1 macrophage polarization, increased the ratio of M1/M2 polarized BMDMs and proliferation/activation of cocultured T cells in vitro, indicating angiogenesis-independent immunomodulatory effects. Suppression of p38 kinase phosphorylation and downstream Creb1/Klf4 activity in BMDMs by regorafenib reversed M2 polarization. Regorafenib enhanced antitumor efficacy of adoptively transferred antigen-specific T cells. Synergistic antitumor efficacy between regorafenib and anti-PD1 was associated with multiple immune-related pathways in the tumor microenvironment. CONCLUSION: Regorafenib may enhance antitumor immunity through modulation of macrophage polarization, independent of its anti-angiogenic effects. Optimization of regorafenib dosage for rational design of combination therapy regimen may improve the therapeutic index in the clinic.

背景：regorafenib 和其他多激酶抑制剂可能会增强抗程序性细胞死亡-1（抗 PD-1）治疗在肝细胞癌（HCC）中的抗肿瘤疗效。除了抗血管生成作用外，其免疫调节作用尚不清楚。

方法：在多个同种异体肝癌模型中进行体内抗肿瘤疗效测试。在体外，用regorafenib 测试小鼠骨髓来源的巨噬细胞（BMDM）对极化的调节作用，并激活共培养的 T 细胞。通过定量逆转录 PCR（RT-PCR）、精氨酸酶活性、流式细胞术和 ELISA 测量 M1/M2 极化的标志物。通过使用 MAPK14 激酶的上游激酶敲低、化学 p38 激酶抑制剂和染色质免疫沉淀，证实了巨噬细胞极化中 p38 激酶和下游 Creb1/Klf4 信号的敲低。

结果：regorafenib（5mg/kg/天，相当于人类临床剂量的一半左右）在体内抑制肿瘤生长和血管生成的作用与 DC-101（抗 VEGFR2 抗体）相似，但产生了更高的 T 细胞激活和 M1 巨噬细胞极化，增加了体外共培养的 BMDM 中 M1/M2 极化的比例和 T 细胞的增殖/激活，表明存在非血管生成的免疫调节作用。regorafenib 抑制 BMDM 中的 p38 激酶磷酸化和下游 Creb1/Klf4 活性，逆转了 M2 极化。regorafenib 增强了过继转移的抗原特异性 T 细胞的抗肿瘤疗效。regorafenib 与抗 PD-1 之间的协同抗肿瘤疗效与肿瘤微环境中的多种免疫相关途径有关。

结论：regorafenib 可能通过调节巨噬细胞极化来增强抗肿瘤免疫，而不依赖于其抗血管生成作用。优化 regorafenib 的剂量以合理设计联合治疗方案可能会提高临床治疗指数。