Periodic Reporting for period 2 - PAVE (A nanovaccine Approach for the treatment of Pancreatic Cancer)
Periodo di rendicontazione: 2021-10-01 al 2024-03-31
Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths by 2030 and represents one of the deadliest cancer types, globally accounting for 4.7% of all deaths for cancer, with a median life expectancy of <1 year for PDAC patients with metastasis. This dismal prognosis can be attributed to late detection, tumour heterogeneity, intrinsic chemoresistance and failure of conventional therapeutic approaches. New approaches, such as immunotherapy still face obstacles in PDAC, as it is an inherently immune-cold tumour and uses various strategies to counteract the effectiveness of immunotherapy, such as a dense stroma that hinders the infiltration of T-cells into the tumour microenvironment (TME), promotes chemoresistance and immune escape, and produces cytokines that support the growth and survival of the tumour cells. Additionally, the translation of immunotherapy into clinical applications is impeded by the inadequacy of effective pre-clinical models. Therefore, the overall aim of PAVE was to establish new strategies and approaches for nanoparticle (NP)-based cancer vaccines, in conjunction with modulation of the TME, for the treatment of PDAC. Our goals were to achieve long-term immune suppression of PDAC, using multicomponent nanovaccines, to produce adequate preclinical models and assays, which will be more relevant for testing these new immunological approaches and to track vaccine biodistribution in vivo. This approach included the establishment of PDAC porcine models that replicate the human immune system, allowing preclinical testing of our innovative NP-based vaccines with multiple, rather than single, encapsulated immunomodulating components. The collaboration between researchers, developers, regulators, and clinicians as well as between industry and academia allowed the development of novel nanovaccine-based immunotherapies with the high promise to prolong survival and quality-of-life of PDAC patients.
The project generated multicomponent nanovaccines targeting tumour associated antigens by encapsulating different peptides (NY-ESO1, MSLN and KRAS), as well as different adjuvants such as the TLR3 agonist Poly(I:C; pIC) and the dual TLR7/8 activator Resiquimod. Covalent attachment of polyethylene glycol chains increased the solubility, bulk amount, and recovery of most peptides. Several of the synthesized peptides were encapsulated into polymer PLGA-NPs, that were coated with chitosan to prevent aggregation and increase the affinity to tumour cells. Nanovaccines were partly labeled with fluorescent dyes, for visual tracking in both in vitro and in vivo assays. These vaccines were designed to stimulate a robust immune response, particularly by activating T-cells against PDAC cells.
Recognizing the limitations of existing preclinical models, PAVE developed new models, including a porcine (pig) model that replicates human PDAC more accurately than mouse models. This "oncopig" with specific genetic mutations relevant to PDAC, provides a valuable tool for testing the efficacy of new therapies. In addition, PAVE succeeded in developing a toolbox of pig-specific immunology reagents and assays including bioactive immune proteins, cloned cytokines and chemokines, and monoclonal antibodies to the proteins and their receptors for the porcine model.
PAVE researchers developed several innovative imaging tools, such as near-infrared fluorescent biosensors, transgenic mice with bioluminescent and fluorescent gene reporters, and optoacoustic imaging techniques. These tools enable for example real-time tracking of vaccine biodistribution, T-cell activation, and tumour progression in vivo, enhancing the ability to monitor treatment responses and thus optimize therapy.
The project made significant progress in understanding the immunosuppressive nature of the PDAC tumour microenvironment (TME). This included the identification of immunoregulatory genes (IRGs) whose knockdown improved T-cell mediated tumor killing, the characterization of the heterogeneity of cancer associated fibroblasts (CAFs) in human PDAC, and the development of a novel platform for constructing immune component-based antigens targeting key PDAC proteins such as MSLN and KRAS.
Research on the role of extracellular vesicles (EVs) in PDAC revealed their strong thrombotic potential, which could contribute to the understanding of the high incidence of thrombosis in PDAC patients. This finding opens the door to use EVs as biomarkers for assessing thrombosis risk in these patients.
PAVE demonstrated that the MSLN-based nanovaccines could effectively reduce tumor growth and metastasis in PDAC mouse models. These nanovaccines further showed potential synergistic effects when combined with other therapies, such as immune checkpoint inhibitors and conventional chemotherapy in human PDAC organoid co-cultures. Given age-related immune decline, the project also investigated whether senolysis before vaccination could enhance immune responses in aged mice and whether there is an age-related difference in immune response to the MSLN nanovaccine. No significant difference was observed between young and old mice in their response.
The project also advanced methods for analyzing tumor heterogeneity using single-cell data, which is crucial for understanding the complex interactions of tumour, immune cells and fibroblasts within the TME and for identifying new therapeutic targets.
Recognizing the limitations of existing preclinical models, PAVE developed new models, including a porcine (pig) model that replicates human PDAC more accurately than mouse models. This "oncopig" with specific genetic mutations relevant to PDAC, provides a valuable tool for testing the efficacy of new therapies. In addition, PAVE succeeded in developing a toolbox of pig-specific immunology reagents and assays including bioactive immune proteins, cloned cytokines and chemokines, and monoclonal antibodies to the proteins and their receptors for the porcine model.
PAVE researchers developed several innovative imaging tools, such as near-infrared fluorescent biosensors, transgenic mice with bioluminescent and fluorescent gene reporters, and optoacoustic imaging techniques. These tools enable for example real-time tracking of vaccine biodistribution, T-cell activation, and tumour progression in vivo, enhancing the ability to monitor treatment responses and thus optimize therapy.
The project made significant progress in understanding the immunosuppressive nature of the PDAC tumour microenvironment (TME). This included the identification of immunoregulatory genes (IRGs) whose knockdown improved T-cell mediated tumor killing, the characterization of the heterogeneity of cancer associated fibroblasts (CAFs) in human PDAC, and the development of a novel platform for constructing immune component-based antigens targeting key PDAC proteins such as MSLN and KRAS.
Research on the role of extracellular vesicles (EVs) in PDAC revealed their strong thrombotic potential, which could contribute to the understanding of the high incidence of thrombosis in PDAC patients. This finding opens the door to use EVs as biomarkers for assessing thrombosis risk in these patients.
PAVE demonstrated that the MSLN-based nanovaccines could effectively reduce tumor growth and metastasis in PDAC mouse models. These nanovaccines further showed potential synergistic effects when combined with other therapies, such as immune checkpoint inhibitors and conventional chemotherapy in human PDAC organoid co-cultures. Given age-related immune decline, the project also investigated whether senolysis before vaccination could enhance immune responses in aged mice and whether there is an age-related difference in immune response to the MSLN nanovaccine. No significant difference was observed between young and old mice in their response.
The project also advanced methods for analyzing tumor heterogeneity using single-cell data, which is crucial for understanding the complex interactions of tumour, immune cells and fibroblasts within the TME and for identifying new therapeutic targets.
Since the mesothelin-based nanovaccine shows a moderate therapeutic effect in a PDAC mouse model, this immunotherapy has the potential of future exploitation in particular combined with targeting IRGs with the high promise to improve therapeutic outcome by prolonging survival and quality-of-life of PDAC patients. The successful generation and characterization of a spontaneous porcine PDAC model, carrying three PDAC relevant genetic modifications, as well as pig PDAC derived organoids that reflect human PDAC are currently the only available non-mouse models for PDAC to assess cancer vaccines at a human scale. The tools are now available as preclinical models for testing novel therapeutics and speed up future clinical translation of PDAC therapies. To evaluate the organoid-killing capacity of PDAC-patient derived antigen-stimulated T-cells against PDAC organoids, a co-culture assay of organoids with immune cells and an analysis platform encompassing the visualization of these co-cultures by live imaging systems was established. The developed algorithm (OrganIDNet) enables in future the systematic evaluation of number, size, health status and eccentricity of the organoids in response to immunotherapy over time. Novel optical imaging protocols using bioluminescence, fluorescent probes and optoacoustic imaging as well as optical reporters as a biosensor, and a transgenic mouse bearing a bioluminescent and a fluorescence gene reporter are now available to monitor the T-cell specific response to therapies. Furthermore, methods were developed and optimized to analyze single cell data, which can now be applied to further research in the oncological field.
All ESRs but one received or will receive their PhD in the near future and have established an active and continuing network of researchers. Thus, PAVE has provided them with an excellent foundation for a science-based career in Europe.
All ESRs but one received or will receive their PhD in the near future and have established an active and continuing network of researchers. Thus, PAVE has provided them with an excellent foundation for a science-based career in Europe.