Immunotherapy represents a revolutionary advancement in cancer treatment, which has traditionally focused on T cells; however, the role of B cells in cancer immunotherapy has gained interest because of their role in antigen presentation, antibody production, and cytokine release. In this study, we examined the role of B cells in previously developed intratumoral MBTA therapy (mannan-BAM, TLR ligands, and anti-CD40 antibody) in murine models of MTT pheochromocytoma. The results indicated that B cells significantly enhance the success of MBTA therapy, with wild-type mice exhibiting a lower tumor incidence and smaller tumors compared with B cell-deficient mice. Increased IL-6 and TNF-alpha levels indicated severe inflammation and a potential cytokine storm in B cell-deficient mice. Neutralization of TNF-alpha ameliorated these complications but resulted in increased tumor recurrence. The results highlight the important role of B cells in enhancing the immune response and maintaining immune homeostasis during MBTA therapy. Our findings offer new insights into improving therapeutic outcomes.

• Keywords
• B cells, cytokine storm, intratumoral immunotherapy, melanoma, pheochromocytoma,
• MeSH
• B-Lymphocytes * immunology   physiology   MeSH
• Pheochromocytoma * immunology   therapy   pathology   MeSH
• Immunotherapy * methods   MeSH
• Interleukin-6 MeSH
• Disease Models, Animal MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Adrenal Gland Neoplasms * immunology   therapy   pathology   MeSH
• Tumor Necrosis Factor-alpha immunology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Interleukin-6 MeSH
• Tumor Necrosis Factor-alpha MeSH

Cancer immunotherapy has shown remarkable clinical progress in recent years. Although age is one of the biggest leading risk factors for cancer development and older adults represent a majority of cancer patients, only a few new cancer immunotherapeutic interventions have been preclinically tested in aged animals. Thus, the lack of preclinical studies focused on age-dependent effect during cancer immunotherapy could lead to different therapeutic outcomes in young and aged animals and future modifications of human clinical trials. Here, we compare the efficacy of previously developed and tested intratumoral immunotherapy, based on the combination of polysaccharide mannan, toll-like receptor ligands, and anti-CD40 antibody (MBTA immunotherapy), in young (6 weeks) and aged (71 weeks) mice bearing experimental pheochromocytoma (PHEO). The presented results point out that despite faster growth of PHEO in aged mice MBTA intratumoral immunotherapy is effective approach without age dependence and could be one of the possible therapeutic interventions to enhance immune response to pheochromocytoma and perhaps other tumor types in aged and young hosts.

• Keywords
• TLR ligands, age, anti-CD40 antibody, intratumoral immunotherapy, pancreatic adenocarcinoma, pheochromocytoma,
• MeSH
• CD40 Antigens MeSH
• Pheochromocytoma * therapy   MeSH
• Immunotherapy methods   MeSH
• Humans MeSH
• Mice MeSH
• Adrenal Gland Neoplasms * therapy   MeSH
• Aged MeSH
• Toll-Like Receptors MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Aged MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• CD40 Antigens MeSH
• Toll-Like Receptors MeSH

Despite constant advances in cancer research, the treatment of pancreatic adenocarcinoma remains extremely challenging. The intratumoral immunotherapy approach that was developed by our research group and was based on a combination of mannan-BAM, TLR ligands, and anti-CD40 antibody (MBTA) showed promising therapeutic effects in various murine tumor models, including a pancreatic adenocarcinoma model (Panc02). However, the efficacy of MBTA therapy in the Panc02 model was negatively correlated with tumor size at the time of therapy initiation. Here, we aimed to further improve the outcome of MBTA therapy in the Panc02 model using the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). The combination of intratumoral MBTA therapy and intraperitoneal administration of DON resulted in the complete elimination of advanced Panc02 subcutaneous tumors (140.8 ± 46.8 mm3) in 50% of treated animals and was followed by development of long-term immune memory. In the bilateral Panc02 subcutaneous tumor model, we observed a significant reduction in tumor growth in both tumors as well as prolonged survival of treated animals. The appropriate timing and method of administration of DON were also addressed to maximize its therapeutic effects and minimize its side effects. In summary, our findings demonstrate that the intraperitoneal application of DON significantly improves the efficacy of intratumoral MBTA therapy in both advanced and bilateral Panc02 subcutaneous tumor murine models.

• Keywords
• Cancer, Glutamine antagonist, Immunotherapy, Intratumoral, Pancreatic adenocarcinoma,
• MeSH
• Adenocarcinoma * drug therapy   MeSH
• Glutamine therapeutic use   MeSH
• Immunotherapy methods   MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Pancreatic Neoplasms * drug therapy   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glutamine MeSH

Pancreatic adenocarcinoma is one of the leading causes of cancer-related deaths, and its therapy remains a challenge. Our proposed therapeutic approach is based on the intratumoral injections of mannan-BAM, toll-like receptor ligands, and anti-CD40 antibody (thus termed MBTA therapy), and has shown promising results in the elimination of subcutaneous murine melanoma, pheochromocytoma, colon carcinoma, and smaller pancreatic adenocarcinoma (Panc02). Here, we tested the short- and long-term effects of MBTA therapy in established subcutaneous Panc02 tumors two times larger than in previous study and bilateral Panc02 models as well as the roles of CD4+ and CD8+ T lymphocytes in this therapy. The MBTA therapy resulted in eradication of 67% of Panc02 tumors with the development of long-term memory as evidenced by the rejection of Panc02 cells after subcutaneous and intracranial transplantations. The initial Panc02 tumor elimination is not dependent on the presence of CD4+ T lymphocytes, although these cells seem to be important in long-term survival and resistance against tumor retransplantation. The resistance was revealed to be antigen-specific due to its inability to reject B16-F10 melanoma cells. In the bilateral Panc02 model, MBTA therapy manifested a lower therapeutic response. Despite numerous combinations of MBTA therapy with other therapeutic approaches, our results show that only simultaneous application of MBTA therapy into both tumors has potential for the treatment of the bilateral Panc02 model.

• Keywords
• Cancer immunotherapy, Checkpoint inhibitors, Mannan, Metastases, Pancreatic adenocarcinoma, TLR ligands,
• MeSH
• Adenocarcinoma immunology   pathology   MeSH
• CD40 Antigens antagonists & inhibitors   MeSH
• Imidazoles pharmacology   MeSH
• Immunotherapy MeSH
• Teichoic Acids pharmacology   MeSH
• Ligands MeSH
• Lipopolysaccharides pharmacology   MeSH
• Mannans pharmacology   MeSH
• Mice MeSH
• Pancreatic Neoplasms immunology   pathology   MeSH
• Poly I-C pharmacology   MeSH
• Antineoplastic Combined Chemotherapy Protocols pharmacology   MeSH
• T-Lymphocytes drug effects   immunology   MeSH
• Toll-Like Receptors MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• CD40 Antigens MeSH
• Imidazoles MeSH
• Teichoic Acids MeSH
• Ligands MeSH
• Lipopolysaccharides MeSH
• lipoteichoic acid MeSH Browser
• Mannans MeSH
• Poly I-C MeSH
• resiquimod MeSH Browser
• Toll-Like Receptors MeSH

There is no doubt that immunotherapy lies in the spotlight of current cancer research and clinical trials. However, there are still limitations in the treatment response in certain types of tumors largely due to the presence of the complex network of immunomodulatory and immunosuppressive pathways. These limitations are not likely to be overcome by current immunotherapeutic options, which often target isolated steps in immune pathways preferentially involved in adaptive immunity. Recently, we have developed an innovative anti-cancer immunotherapeutic strategy that initially elicits a strong innate immune response with subsequent activation of adaptive immunity in mouse models. Robust primary innate immune response against tumor cells is induced by toll-like receptor ligands and anti-CD40 agonistic antibodies combined with the phagocytosis-stimulating ligand mannan, anchored to a tumor cell membrane by biocompatible anchor for membrane. This immunotherapeutic approach results in a dramatic therapeutic response in large established murine subcutaneous tumors including melanoma, sarcoma, pancreatic adenocarcinoma, and pheochromocytoma. Additionally, eradication of metastases and/or long-lasting resistance to subsequent re-challenge with tumor cells was also accomplished. Current and future advantages of this immunotherapeutic approach and its possible combinations with other available therapies are discussed in this review.

• Keywords
• Adaptive immunity, Cancer, Immune response, Immunotherapy, Innate immunity,
• MeSH
• Adaptive Immunity MeSH
• Phagocytosis drug effects   immunology   MeSH
• Immune System immunology   metabolism   MeSH
• Immunomodulation MeSH
• Immunotherapy * methods   MeSH
• Combined Modality Therapy MeSH
• Humans MeSH
• Ligands MeSH
• Tumor Microenvironment drug effects   genetics   immunology   MeSH
• Neoplasms etiology   metabolism   pathology   therapy   MeSH
• Immunity, Innate MeSH
• Antineoplastic Agents, Immunological pharmacology   therapeutic use   MeSH
• Toll-Like Receptors metabolism   MeSH
• Treatment Outcome MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Ligands MeSH
• Antineoplastic Agents, Immunological MeSH
• Toll-Like Receptors MeSH

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.

• Keywords
• T cell, adaptive immunity, immunotherapy, innate immunity, metastatic, neutrophil, paraganglioma, pathogen-associated molecular patterns, pheochromocytoma, toll-like receptor,
• Publication type
• Journal Article MeSH

BACKGROUND: Using killed microorganisms or their parts to stimulate immunity for cancer treatment dates back to the end of 19th century. Since then, it undergone considerable development. Our novel approach binds ligands to the tumor cell surface, which stimulates tumor phagocytosis. The therapeutic effect is further amplified by simultaneous application of agonists of Toll-like receptors. We searched for ligands that induce both a strong therapeutic effect and are safe for humans. METHODS: B16-F10 murine melanoma model was used. For the stimulation of phagocytosis, mannan or N-formyl-methionyl-leucyl-phenylalanine, was covalently bound to tumor cells or attached using hydrophobic anchor. The following agonists of Toll-like receptors were studied: monophosphoryl lipid A (MPLA), imiquimod (R-837), resiquimod (R-848), poly(I:C), and heat killed Listeria monocytogenes. RESULTS: R-848 proved to be the most suitable Toll-like receptor agonist for our novel immunotherapeutic approach. In combination with covalently bound mannan, R-848 significantly reduced tumor growth. Adding poly(I:C) and L. monocytogenes resulted in complete recovery in 83% of mice and in their protection from the re-transplantation of melanoma cells. CONCLUSION: An efficient cancer treatment results from the combination of Toll-like receptor agonists and phagocytosis stimulating ligands bound to the tumor cells.

• Keywords
• Cancer immunotherapy, Innate immunity, Mannan, Melanoma, Neutrophils, Phagocytosis, Resiquimod,
• MeSH
• Cytokines metabolism   MeSH
• Phagocytosis MeSH
• Imidazoles pharmacology   MeSH
• Immunotherapy * methods   MeSH
• Neutrophil Infiltration immunology   MeSH
• Ligands MeSH
• Mannans immunology   MeSH
• Melanoma, Experimental MeSH
• Disease Models, Animal MeSH
• Mice MeSH
• Neoplasms immunology   metabolism   pathology   therapy   MeSH
• Neutrophils immunology   metabolism   MeSH
• Poly I-C immunology   MeSH
• Immunity, Innate * MeSH
• Respiratory Burst immunology   MeSH
• Toll-Like Receptors agonists   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines MeSH
• Imidazoles MeSH
• Ligands MeSH
• Mannans MeSH
• Poly I-C MeSH
• resiquimod MeSH Browser
• Toll-Like Receptors MeSH