Systemic Immune Response in Murine Bilateral Pheochromocytoma Model During Immunotherapy Based on a Combination of Mannan-BAM, TLR Ligands and Anti-CD40 Antibodies (MBTA Therapy)

Abstract Immunotherapy has become an essential component of cancer treatment, however, a majority of patients with solid metastatic cancers, such as pheochromocytoma (PHEO), do not respond to this type of therapy. Recently, we developed an intratumoral (i.t.) immunotherapy based on the unique combination of TLR ligands, anti-CD40 antibodies, and mannan, which is artificially bound to tumor cells via an anchor (MBTA therapy). This therapy resulted in the complete eradication of aggressive subcutaneous PHEO in 67% of mice and demonstrated a systemic antitumor immune response and regression of non-treated lesions in the metastatic model (1). To further evaluate this systemic effect generated during MBTA therapy, we established a murine bilateral PHEO model, where MBTA therapy was i.t. injected into one tumor, and the distant (non-treated) tumor was monitored for changes in size and immune cell infiltration. The growth of both MBTA-treated and distant tumors was reduced compared to that of the control. Interestingly, survival of the MBTA-treated mice was twice as long compared to the control mice. Moreover, we have made several unique observations during the experiments which were focused on the tumor microenvironment. Flow cytometry analysis revealed the ability of MBTA therapy to significantly increase the infiltration of innate immune cells (monocytes, DCs, macrophages, NK cells) not only in MBTA-treated tumors, but also in distal tumors, despite the fact that MBTA therapy was designed to elicit only local inflammation. An analysis of the macrophage phenotype revealed a switch from protumor M2 to antitumor M1 macrophages in both tumors during the entire MBTA therapy treatment. Analysis of splenic adaptive immune cells revealed that naïve CD4+ or CD8+ T cells differentiated into central memory cells and effector memory cells. CD4+ and CD8+ T cells were elevated in MBTA-treated and distant tumors with a significantly higher frequency of CD8+ effector memory T cells. Moreover, the adoptive transfer of CD4+ and CD8+ T cells revealed that immune memory, after tumor rechallenging, was driven by CD4+ T cells. Collectively, these results illustrate the ability of MBTA therapy to activate both parts of the immune system and render a systemic antitumor response against non-treated metastases. We believe that our results could lead to the use of MBTA therapy in patients with aggressive, metastatic lesions. Reference: Caisova et al., Cancers (Basel), 2019. 11(5).