The Significant Reduction or Complete Eradication of Subcutaneous and Metastatic Lesions in a Pheochromocytoma Mouse Model after Immunotherapy Using Mannan-BAM, TLR Ligands, and Anti-CD40

Abstract

Therapeutic options for metastatic pheochromocytoma/paraganglioma (PHEO/PGL) are limited. Here, we tested an immunotherapeutic approach based on intratumoral injections of mannan-BAM with toll-like receptor ligands into subcutaneous PHEO in a mouse model. This therapy elicited a strong innate immunity-mediated antitumor response and resulted in a significantly lower PHEO volume compared to the phosphate buffered saline (PBS)-treated group and in a significant improvement in mice survival. The cytotoxic effect of neutrophils, as innate immune cells predominantly infiltrating treated tumors, was verified in vitro. Moreover, the combination of mannan-BAM and toll-like receptor ligands with agonistic anti-CD40 was associated with increased mice survival. Subsequent tumor re-challenge also supported adaptive immunity activation, reflected primarily by long-term tumor-specific memory. These results were further verified in metastatic PHEO, where the intratumoral injections of mannan-BAM, toll-like receptor ligands, and anti-CD40 into subcutaneous tumors resulted in significantly less intense bioluminescence signals of liver metastatic lesions induced by tail vein injection compared to the PBS-treated group. Subsequent experiments focusing on the depletion of T cell subpopulations confirmed the crucial role of CD8+ T cells in inhibition of bioluminescence signal intensity of liver metastatic lesions. These data call for a new therapeutic approach in patients with metastatic PHEO/PGL using immunotherapy that initially activates innate immunity followed by an adaptive immune response.

Induction of Immune Response against Metastatic Tumors via Vaccination of Mannan‐BAM, TLR Ligands, and Anti‐CD40 Antibody (MBTA)

“…Previous MBTA investigations demonstrated that in situ injection of MBTA induced potent innate immune responses against vaccinated tumors. [ 8,9 ] More recently, in a pheochromocytoma mouse model, Caisova et al. demonstrated that in situ injection of MBTA resulted in lower tumor burden of metastatic organ lesions when compared to PBS‐treated control mice and significantly prolonged survival.…”

“…Recently, a promising immunotherapeutic strategy consisting of a combination of mannan, a polysaccharide derived from Saccharomyces cerevisiae , toll‐like receptor (TLR) ligands, and agonistic anti‐CD40‐monoclonal antibody (abbreviated as MBTA) demonstrated potent antitumor responses in several murine cancer models when injected intratumorally (in situ). [ 8,9 ] Mechanistically, mannan serves as a phagocytosis‐stimulating ligand when conjugated to Biocompatible Anchor for Cell Membrane (BAM). [ 10,11 ] The linkage of mannan to BAM (Mannan‐BAM) facilitates anchoring of mannan to cell membranes via BAM's hydrophobic oleyl group and subsequently exploits mannan recognition by pattern recognition receptors, leading to complement activation and opsonophagocytosis of tumor cells.…”

“…To bolster mannan‐BAM's inductive effect on innate immune cells, three TLR ligands (lipoteichoic acid (LTA), polyinosinic‐polycytidylic acid poly(I:C), and resiquimod (R‐848)) and immunostimulatory anti‐CD40‐mAb were incorporated as adjuvants. [ 8,9,11,13–15 ] LTA from Bacillus subtilis activates TLR2 mediated inflammatory pathways known to increase TNFα secretion. [ 16,17 ] Poly(I:C), a synthetic analog of viral dsRNA, activates TLR3 mediated signaling previously demonstrated to activate antigen‐presenting cells (APCs), modulate the phenotype of tumor associated macrophages to more immunosupportive phenotypes, and induce tumor cell apoptosis.…”

“…Prior MBTA studies demonstrated striking efficacy in regressing primary tumors, injected with MBTA. [ 8,9 ] However, the efficacy and characterization of the immune response generated against distal, untreated tumors was not explored. Additionally, previous experimental mouse models of MBTA have relied on the in situ injection of MBTA to generate an antitumor immune response.…”

“…Additionally, previous experimental mouse models of MBTA have relied on the in situ injection of MBTA to generate an antitumor immune response. [ 8,9 ] Such therapeutic delivery strategy has demonstrated efficacy in improving tumor growth control and overall mouse survival. However, a significant limitation of the in situ injection strategy is its reliance on an anatomically accessible tumor to inject MBTA.…”