Periodic Reporting for period 5 - STIMUNO (Searching for novel strategies improving cancer immunotherapy)
Période du rapport: 2024-12-01 au 2025-12-31
Cancer immunotherapy has recently become an important component of the combination treatment schedules for patients with multiple types of cancer. However, despite spectacular clinical responses in some patients, the immunosuppressive tumor microenvironment (TME) constitutes a key barrier to effective immunotherapy. Therefore, the identification of immunosuppressive mechanisms within TME which have not been studied so far becomes the pressing subject in the field of anti-tumor immunotherapy.
The overall objective of this project is to explore novel metabolic pathways involved in the regulation of antitumor immune response. Specifically, we concentrated on amino acid metabolism in both hematological and solid tumor models. Within the tumor microenvironment, we have discovered a new metabolite, ammonia, with a robust ability to inhibit the activity of immune cells and their potential to kill target tumor cells. The knowledge gained so far during the implementation of this project can potentially help to design novel therapeutic approaches that could further increase response rates in cancer patients.
The overall objective of this project is to explore novel metabolic pathways involved in the regulation of antitumor immune response. Specifically, we concentrated on amino acid metabolism in both hematological and solid tumor models. Within the tumor microenvironment, we have discovered a new metabolite, ammonia, with a robust ability to inhibit the activity of immune cells and their potential to kill target tumor cells. The knowledge gained so far during the implementation of this project can potentially help to design novel therapeutic approaches that could further increase response rates in cancer patients.
We identified ammonia as a byproduct of amino acid metabolism as an important factor limiting the activity of effector immune cells. We have documented that ammonia can be accumulated in tumor cell cultures and tumor interstitial fluid (TIF) isolated from mice inoculated with tumor cell lines. We have established a method of isolation of TIF from murine tumors that enables the analysis of tumor metabolome. To elucidate the mechanisms responsible for the immunosuppressive activity of ammonia, we optimized a vast array of methods to study the activity of NK cells and CAR-T cells. We observed that ammonia inhibits the natural cytotoxicity of NK cells, as well as CAR-mediated killing. To further identify the mechanisms of inhibition, we optimized and employed methods to study degranulation, cytokines production, and synapse formation. We determined that ammonia, as a lysosomotropic agent, impairs the process of perforin production and maturation and, in this mechanism, inhibits the cytotoxic potential of effector cells. Altogether, we have identified a unique, novel and never described before mechanism by which ammonia interferes with cytotoxic activity of T cells and NK cells. The experiments on the role and mechanism of impairment of cytotoxic cells by ammonia were published in Cancer Research journal (doi: 10.1158/0008-5472.CAN-24-0749).
In the screening experiments, we also observed that liver cancer cell lines produce low amounts of ammonia. To better mimic the tumor microenvironment and ammonia distribution within the liver, we optimized more advanced orthotopic models of hepatocellular carcinoma (HCC) in xenograft model of NSG mice with tumor cells administered directly into the portal vein. In these orthotopic in vivo models of HCC, we observed no accumulation of ammonia in TIF. Therefore, we subsequently selected a liver cancer model characterized by low ammonia concentrations to determine the efficacy of CAR-based immunotherapy. Subsequently, we developed PD-L1-CAR T cell therapy administered through the splenic vein to directly target liver neoplasms and reshape TME. PD-L1-CAR T cells effectively eliminated liver cancer cell lines in vitro, however, systemic delivery in vivo resulted in lethal toxicity. To improve the safety profile, liver locoregional administration was employed which helped to avoid major side effects, preserve organ integrity, and maintain anticancer efficacy. Additionally, PD-L1-CAR T cells demonstrated high efficacy against patient-derived liver tumor cultures from diverse origins. Altogether, we showed that locoregional administration of PD-L1-CAR T cell is effective against liver cancer.
Using the optimized method of TIF isolation, we also quantified other immunosuppressive metabolites in TME. We demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo. We also observed that NK cells were the most susceptible to hydrogen peroxide and identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. Finally, we generated PD-L1–CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells. These experiments were published in Cancer Immunology Research journal (doi: 10.1158/2326-6066.CIR-20-1023).
In the screening experiments, we also observed that liver cancer cell lines produce low amounts of ammonia. To better mimic the tumor microenvironment and ammonia distribution within the liver, we optimized more advanced orthotopic models of hepatocellular carcinoma (HCC) in xenograft model of NSG mice with tumor cells administered directly into the portal vein. In these orthotopic in vivo models of HCC, we observed no accumulation of ammonia in TIF. Therefore, we subsequently selected a liver cancer model characterized by low ammonia concentrations to determine the efficacy of CAR-based immunotherapy. Subsequently, we developed PD-L1-CAR T cell therapy administered through the splenic vein to directly target liver neoplasms and reshape TME. PD-L1-CAR T cells effectively eliminated liver cancer cell lines in vitro, however, systemic delivery in vivo resulted in lethal toxicity. To improve the safety profile, liver locoregional administration was employed which helped to avoid major side effects, preserve organ integrity, and maintain anticancer efficacy. Additionally, PD-L1-CAR T cells demonstrated high efficacy against patient-derived liver tumor cultures from diverse origins. Altogether, we showed that locoregional administration of PD-L1-CAR T cell is effective against liver cancer.
Using the optimized method of TIF isolation, we also quantified other immunosuppressive metabolites in TME. We demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo. We also observed that NK cells were the most susceptible to hydrogen peroxide and identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. Finally, we generated PD-L1–CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells. These experiments were published in Cancer Immunology Research journal (doi: 10.1158/2326-6066.CIR-20-1023).
During the project implementation, several important technological advances have been achieved. They not only contributed to the effective implementation of the ERC Starting Grant project, but also significantly improved scientific capacity of the entire team at the Department of Immunology, MMRI, PAS, Warsaw, Poland. Specifically, a platform for the assessment of influence of immunosuppressive TME on antitumor activity of various immunotherapies was developed. It has played a crucial role in the development and evaluation of therapies based on CAR-T cells, BiTes, NK cells and allowed for studying their efficacy, safety, and mechanisms of action. By using this platform, we have identified ammonia as an immunoinhibitory metabolite within the tumor microenvironment. Ammonia had not been previously recognized for its impact on the immune system; however, at the same time as we were implementing our project, an increasing number of studies have highlighted its modulatory role. In this context, our research aligns with recent discoveries and is in line with the very latest advancements in the field. We have identified a pivotal connection between metabolism and immunotherapy efficacy. By showing that ammonia not only promotes tumor growth as a nutrient source but also hinders immune responses, we underscore the importance of considering metabolic by-products in the context of immunotherapy. This dual role of ammonia as both a tumor promoter and an inhibitor of immune function represents a crucial advancement in understanding the biology of the cancer. The findings suggest that strategies aimed at reducing ammonia levels or counteracting its effects in the tumor microenvironment may enhance the effectiveness of existing NK and CAR T-cell therapies. This novel perspective could lead to innovative therapeutic approaches, combining metabolic interventions with immunotherapy to overcome current limitations.