Silva Priscila, Silva Gabrielli Novaes, Melo Filipe Menegatti, de Amat Herbozo Carolina, Sellani Tarciso Almeida, Tomaz Samanta Lopes, De Melo Amanda Campelo L, Da Silva Larissa Reis, Berzaghi Rodrigo, Marcondes Marcelo F M, Bronze Fellipe, Paschoalin Thaysa, Glezer Isaias, Carmona Adriana K, Pereira Felipe Valença, Rodrigues Elaine Guadelupe
Department of Microbiology, Immunology, and Parasitology, Paulista School of Medicine, Federal University of São Paulo (EPM-UNIFESP), São Paulo, Brazil.
Goethe University Frankfurt, Faculty of Medicine, Institute of Clinical Pharmacology, Frankfurt/Main, Germany.
Front Immunol. 2025 Sep 12;16:1630886. doi: 10.3389/fimmu.2025.1630886. eCollection 2025.
Immunosuppressive factors within the tumor microenvironment hinder effective antitumor immune responses and limit the efficacy of current immunotherapies. Immunomodulators offer an alternative by activating immune effectors. Proteases from various sources used as cancer therapy adjuvants have shown promise in inhibiting tumor growth. Our previous work showed that the bacterial metalloprotease arazyme has a strong in vivo antimetastatic effect in the B16F10-Nex2 murine melanoma model. Interestingly, heat-inactivated arazyme also exhibited antitumor properties dependent on an intact adaptive immune response, highlighting its immunomodulatory role. To assess whether this effect is unique to arazyme, we examined another bacterial metalloprotease, Oligopeptidase A (OpdA).
OpdA was produced and purified. Endotoxin levels were measured. C57BL/6 mice received intravenous B16F10-Nex2 cells, followed by treatments with either active or heat-inactivated OpdA. Pulmonary nodules were counted. Immune cells involved in the response were characterized using FACS and depletion experiments. Cytokines were measured by ELISA and intracellular cytokine analysis. OpdA receptor activation was studied in bone marrow-derived cells from knockout and wild-type mice using inhibitors.
Heat-inactivated OpdA significantly reduced metastasis, dependent on tumor-specific CD4+ and CD8+ T cells and IFN-γ, both locally and systemically, with decreased IL-10 levels suggesting a proinflammatory environment. Treatment increased secretion of nitric oxide, IL-12p40, and TNF-α from bone marrow cells via enzymatic activity, involving MyD88/TRIF and MAPK pathways. Conclusion: OpdA shows potential as a tumor vaccine adjuvant, promoting antigen presentation and tumor-specific immune responses.
肿瘤微环境中的免疫抑制因子会阻碍有效的抗肿瘤免疫反应，并限制当前免疫疗法的疗效。免疫调节剂通过激活免疫效应器提供了一种替代方案。来自各种来源的蛋白酶用作癌症治疗佐剂已显示出抑制肿瘤生长的前景。我们之前的研究表明，细菌金属蛋白酶阿雷酶在B16F10-Nex2小鼠黑色素瘤模型中具有很强的体内抗转移作用。有趣的是，热灭活的阿雷酶也表现出依赖于完整适应性免疫反应的抗肿瘤特性，突出了其免疫调节作用。为了评估这种效应是否是阿雷酶所特有的，我们研究了另一种细菌金属蛋白酶，寡肽酶A（OpdA）。
生产并纯化OpdA。测量内毒素水平。C57BL/6小鼠静脉注射B16F10-Nex2细胞，随后用活性或热灭活的OpdA进行治疗。对肺结节进行计数。使用流式细胞术和清除实验对参与反应的免疫细胞进行表征。通过酶联免疫吸附测定法和细胞内细胞因子分析测量细胞因子。使用抑制剂在基因敲除和野生型小鼠的骨髓来源细胞中研究OpdA受体激活。
热灭活的OpdA显著减少转移，这依赖于肿瘤特异性CD4+和CD8+T细胞以及干扰素-γ，在局部和全身均如此，白细胞介素-10水平降低表明存在促炎环境。治疗通过酶活性增加了骨髓细胞中一氧化氮、白细胞介素-12p40和肿瘤坏死因子-α的分泌，涉及髓样分化因子88/ Toll样受体接头蛋白诱导干扰素β和丝裂原活化蛋白激酶途径。结论：OpdA显示出作为肿瘤疫苗佐剂的潜力，可促进抗原呈递和肿瘤特异性免疫反应。
