THE IMMUNOMODULATORY AND ANTITUMOR PROPERTIES OF THE BACTERIAL METALLOPROTEASE OLIGOPEPTIDASE A (OpdA) ARE MEDIATED BY TLR4/MyD88/TRIF AND MAPK SIGNALING PATHWAYS

Priscila Silva^1,2Gabrielli Novaes Silva¹Filipe Menegatti Melo^1,3Carolina de Amat Herbozo¹Tarciso Almeida Sellani^1,4Samanta Lopez Tomaz^1,5Amanda Campelo L. de Melo^1,6Larissa Rodrigues Silva¹Rodrigo Berzaghi^1,7Marcelo F.M. Marcondes⁸Fellipe Bronze⁸Thaysa Paschoalin⁸Isaias Glezer⁹Adriana K. Carmona⁸Felipe Valença Pereira^1,10*Elaine Guadelupe Rodrigues^1*

• ¹Department of Microbiology, Immunology and Parasitology, Paulista School of Medicine, Federal University of São Paulo, São Paulo, Brazil
• ²Goethe University Frankfurt, Faculty of Medicine, Institute of Clinical Pharmacology,, Frankfurt, Germany
• ³Howard Hughes Medical Institute at University of California, California, United States
• ⁴Glaxosmithkline Brasil, Oncohematology, São Paulo, Brazil
• ⁵Sirio-Libanes Hospital, São Paulo, Brazil
• ⁶Oswaldo Cruz Foundation, Ceará, Brazil
• ⁷Department of Clinical Medicine, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø, Norway
• ⁸Department of Biophysics, Paulista School of Medicine, Federal University of São Paulo (EPM-UNIFESP), São Paulo, Brazil, São Paulo, Brazil
• ⁹Department of Biochemistry, Paulista School of Medicine, Federal University of São Paulo (EPM-UNIFESP), São Paulo, Brazil
• ¹⁰Department of Immunology and Microbiology, University of Colorado, Anschutz Medical Campus, Colorado, United States

Immunosuppressive factors in the tumor microenvironment hinder the effectiveness of the antitumor immune response and decrease the success of current antitumor immunotherapies. Immunomodulators provide an appealing alternative to counteract this immunosuppression by activating immune response effectors. Various proteases, isolated from different sources and used as therapeutic adjuvants in cancer treatment, have shown promising results in inhibiting tumor growth. Previous results from our group indicate that the proteolytically active bacterial metalloprotease arazyme has a strong in vivo antimetastatic effect in the B16F10-Nex2 murine melanoma model. Surprisingly, even heat-inactivated arazyme exhibited an antimetastatic effect that depended on an intact adaptive immune response, indicating its immunomodulatory properties. To determine whether this effect is unique to arazyme, we analyzed the potential antitumor and immunomodulatory effects of another bacterial metalloprotease, Oligopeptidase A (OpdA). We found that heat-inactivated OpdA has a significant antimetastatic effect in the B16F10-Nex2 melanoma murine model. This effect depends on tumor-specific CD4+ and CD8+ T lymphocytes, as well as local and systemic IFN-γ. It is accompanied by a notable decrease in IL-10 levels, suggesting that OpdA treatment induces a proinflammatory environment. Additionally, treatment with either active or heat-inactivated OpdA increases the secretion of nitric oxide, IL-12p40, and TNF-α— proinflammatory cytokines—from bone marrow-derived cells. Importantly, this immunomodulatory effect operates independently of OpdA's enzymatic activity and is mediated through the TLR4/MyD88/TRIF and MAPK signaling pathways. In conclusion, these results suggest that OpdA could serve as an adjuvant in tumor vaccines, enhancing antigen presentation and fostering a protective tumor-specific immune response.