Polymeric Nanoparticles for Oral Delivery of a Chimeric Protein as a Vaccination Approach for the Treatment of Glypican-1-expressing Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal forms of cancer, characterized by strong resistance to conventional therapies and a poor prognosis, with frequent relapse and metastasis even after surgical resection and/or standard chemotherapy. Cancer vaccines are emerging as innovative immunotherapeutic approaches, particularly when proposed as adjuvants to be combined with existing treatments. Although vaccines are traditionally administered via parenteral routes, oral delivery offers several advantages for both patients and healthcare systems. Therefore, the present thesis focuses on the development of an oral (OS) nanotechnology-based vaccine platform as an adjuvant therapy for PDAC. The proteoglycan Glypican-1 (GPC1) was selected as the TAA for the development of the protein-based cancer vaccine. Its expression was confirmed in the human PDAC cell line BxPC3 by different techniques. In addition, we selected flagellin (FL) from Salmonella typhimurium as an adjuvant component to enhance vaccine immunogenicity. Therefore, we designed the chimeric protein GPC1-FL and expressed and purified it from Chinese Hamster Ovary cells. We evaluated its stability in PBS at 37 °C, and in simulated gastric fluid (SGF) and simulated intestinal fluids (SIF) buffers that, as expected, was very low. To overcome the limitations of OS delivery, in particular the harsh gastrointestinal environment and the need to target the intestinal immune system, we synthesized PLGA/PVA NPs using a microfluidic nanoprecipitation method, and we characterized them by dynamic light scattering (DLS), confirming their stability over time and at physiological temperature in both SGF and SIF. Furthermore, we demonstrated the NP biocompatibility with the in vitro models of the intestinal epithelium (Caco-2 cells) and antigen-presenting cells (RAW 264.7). In addition, we performed internalization studies using BSA-FITC as a model protein, showing that PLGA/PVA NPs significantly improved its cellular uptake by Caco-2 compared to the free form. Given their stability, biocompatibility, and enhanced internalization capacity, we tested the immunogenicity and the effect on tumor growth of the GPC1-FL-loaded NPs in vivo using a mouse model of PDAC expressing the human GPC1. We demonstrated that the chimeric GPC1-FL protein was immunogenic, eliciting a strong and specific IgG response after subcutaneous administration. This response was further enhanced when the protein was delivered via PLGA/PVA NPs, indicating that NP encapsulation improves antigen presentation and vaccine efficacy. Following OS administration, the free protein did not elicit any detectable IgG response, whereas GPC1-FL-loaded NPs induced a significant, though lower, antibody response compared to subcutaneous vaccination. Finally, mice receiving SC GPC1-FL-loaded PLGA/PVA NPs showed a significantly longer survival time compared to other experimental groups, whereas the immune response generated by OS administration was insufficient to improve survival outcomes. In conclusion, this study developed a novel nanoplatform for SC and OS vaccination against PDAC. Through a multidisciplinary approach encompassing antigen selection (GPC1), production of the immunogenic chimeric protein GPC1-FL, synthesis of PLGA/PVA NPs, and both in vitro and in vivo validation, the project demonstrated that PLGA/PVA NPs effectively enhance the anti-GPC1 immune response, particularly following SC administration, leading to a significant increase in survival in a PDAC mouse model. Although the OS route elicited a weaker immune response, insufficient to delay tumor progression, the results highlight the potential of PLGA/PVA NPs as a safe and effective platform for mucosal vaccination. Further optimization of dose, administration scheduling, or formulation may be necessary for the oral administration to achieve an efficacy comparable to that of the subcutaneous one.