基于生物纳米颗粒的疫苗和检查点阻断刺激的适应性抗肿瘤免疫反应。

BACKGROUND: Interactions between tumor and microenvironment determine individual response to immunotherapy. Triple negative breast cancer (TNBC) and hepatocellular carcinoma (HCC) have exhibited suboptimal responses to immune checkpoint inhibitors (ICIs). Aspartate β-hydroxylase (ASPH), an oncofetal protein and tumor associated antigen (TAA), is a potential target for immunotherapy. METHODS: Subcutaneous HCC and orthotopic TNBC murine models were established in immunocompetent BALB/c mice with injection of BNL-T3 and 4 T1 cells, respectively. Immunohistochemistry, immunofluorescence, H&E, flow cytometry, ELISA and in vitro cytotoxicity assays were performed. RESULTS: The ASPH-MYC signaling cascade upregulates PD-L1 expression on breast and liver tumor cells. A bio-nanoparticle based λ phage vaccine targeting ASPH was administrated to mice harboring syngeneic HCC or TNBC tumors, either alone or in combination with PD-1 blockade. In control, autocrine chemokine ligand 13 (CXCL13)-C-X-C chemokine receptor type 5 (CXCR5) axis promoted tumor development and progression in HCC and TNBC. Interactions between PD-L1 cancer cells and PD-1 T cells resulted in T cell exhaustion and apoptosis, causing immune evasion of cancer cells. In contrast, combination therapy (Vaccine+PD-1 inhibitor) significantly suppressed primary hepatic or mammary tumor growth (with distant pulmonary metastases in TNBC). Adaptive immune responses were attributed to expansion of activated CD4 T helper type 1 (Th1)/CD8 cytotoxic T cells (CTLs) that displayed enhanced effector functions, and maturation of plasma cells that secreted high titers of ASPH-specific antibody. Combination therapy significantly reduced tumor infiltration of immunosuppressive CD4/CD25/FOXP3 Tregs. When the PD-1/PD-L1 signal was inhibited, CXCL13 produced by ASPH cancer cells recruited CXCR5/CD8 T lymphocytes to tertiary lymphoid structures (TLSs), comprising effector and memory CTLs, T follicular helper cells, B cell germinal center, and follicular dendritic cells. TLSs facilitate activation and maturation of DCs and actively recruit immune subsets to tumor microenvironment. These CTLs secreted CXCL13 to recruit more CXCR5 immune cells and to lyse CXCR5 cancer cells. Upon combination treatment, formation of TLSs predicts sensitivity to ICI blockade. Combination therapy substantially prolonged overall survival of mice with HCC or TNBC. CONCLUSIONS: Synergistic antitumor efficacy attributable to a λ phage vaccine specifically targeting ASPH, an ideal TAA, combined with ICIs, inhibits tumor growth and progression of TNBC and HCC.

背景：肿瘤与微环境的相互作用决定了个体对免疫疗法的反应。三阴性乳腺癌（TNBC）和肝细胞癌（HCC）对免疫检查点抑制剂（ICIs）的反应不佳。天门冬氨酸β-羟化酶（ASPH）是一种癌胚蛋白和肿瘤相关抗原（TAA），是免疫治疗的潜在靶点。

方法：在免疫功能正常的 BALB/c 小鼠中分别通过注射 BNL-T3 和 4T1 细胞建立皮下 HCC 和原位 TNBC 小鼠模型。进行免疫组织化学、免疫荧光、H&E、流式细胞术、ELISA 和体外细胞毒性测定。

结果：ASPH-MYC 信号级联反应上调了乳腺癌和肝癌肿瘤细胞上的 PD-L1 表达。一种针对 ASPH 的基于生物纳米颗粒的 λ噬菌体疫苗被施用于携带同源 HCC 或 TNBC 肿瘤的小鼠，单独或与 PD-1 阻断联合使用。在对照中，自分泌趋化因子配体 13（CXCL13）-C-X-C 趋化因子受体 5（CXCR5）轴促进了 HCC 和 TNBC 中的肿瘤发生和进展。PD-L1 癌细胞与 PD-1 T 细胞之间的相互作用导致 T 细胞耗竭和凋亡，从而使癌细胞逃避免疫。相比之下，联合治疗（疫苗+PD-1 抑制剂）显著抑制了原发性肝或乳腺肿瘤的生长（在 TNBC 中有远处肺转移）。适应性免疫反应归因于激活的 CD4 T 辅助 1 型（Th1）/CD8 细胞毒性 T 细胞（CTL）的扩增，这些细胞显示出增强的效应功能，以及浆细胞的成熟，浆细胞分泌高滴度的 ASPH 特异性抗体。联合治疗显著减少了肿瘤浸润的免疫抑制性 CD4/CD25/FOXP3 Tregs。当抑制 PD-1/PD-L1 信号时，ASPH 癌细胞产生的 CXCL13 招募了 CXCR5/CD8 T 淋巴细胞到三级淋巴结构（TLSs）中，其中包括效应和记忆 CTL、滤泡辅助 T 细胞、B 细胞生发中心和滤泡树突状细胞。TLSs 促进了 DC 的激活和成熟，并主动将免疫细胞亚群招募到肿瘤微环境中。这些 CTLs 分泌 CXCL13 以招募更多的 CXCR5 免疫细胞并裂解 CXCR5 癌细胞。在联合治疗时，TLS 的形成预测了对 ICI 阻断的敏感性。联合治疗显著延长了 HCC 或 TNBC 小鼠的总生存期。

结论：特异性针对 ASPH（理想的 TAA）的 λ 噬菌体疫苗与 ICIs 的协同抗肿瘤疗效抑制了 TNBC 和 HCC 的肿瘤生长和进展。