Nanoparticle-based strategies in cancer immunology: from design to clinical implementation

This Ph.D. thesis describes the synthesis, characterization, and application of several nanoparticles in cancer immunology. Chapter 1 provides a general introduction to the relevance of nanotechnology in the biomedical field in the past few years. In Chapter 2, mixed self-assembled monolayers were prepared on both gold nanoparticles and flat surfaces with the objective of examining the impact of nanoparticle coating surface disorder and surface charge density on the adsorption of proteins and cellular uptake by immune system cells. The introduction of surface disorder results in diminished protein adsorption and reduced cellular uptake by phagocytes. Conversely, exploring surface charge density, neutral coatings present higher hydrophilicity, and reduced protein adsorption and cellular uptake. Furthermore, the impact of the protein corona was assessed on AuNP internalization suggesting that the properties of the coating primarily determines the biological interactions. The results obtained during this chapter highlight the possibility of engineering nanoparticle coatings to modulate the interactions with immune cells by introducing small variations in the coating composition. Later, AuNPs passivated with mixed self-assembled monolayers were employed to covalently functionalize gold nanoparticles with a cancer antigen in Chapter 3. While MUC1 stands out as a widely acknowledged cancer antigen, the pursuit of clinically relevant immunotherapy has not yet been accomplished. MUC1 glycopeptides, often elicit only a weak immune response due to their autoantigenic nature. To address this limitation, synthetic antigens with precise modifications were engineered, aiming to mimic the conformational dynamics of natural antigens and enhance binding affinity to anti-MUC1 antibodies. As a proof of concept, a glycopeptide containing the noncanonical amino acid (2S,3R)-3-hydroxynorvaline was developed, effectively reproducing the natural threonine-derived antigen. Conjugation of the antigen to AuNPs promoted the generation of specific anti-MUC1 IgG antibodies in BALB/c mice, demonstrating efficacy comparable to the natural derivative. Notably, these antibodies exhibited cross-reactivity, selectively targeting human breast cancer cells. Chapter 4 focused on the exploitation of circulating tumor-associated autoantibodies, specifically targeting tumor-associated MUC1 as potential biomarkers for pancreatic cancer detection. While previous efforts primarily used unglycosylated models, this study introduced a structure-based approach involving artificial O-GalNAc glycopeptides as TA-MUC1 mimics. These glycopeptides, designed through structural analysis and synthesized in a small library, aim to detect distinct autoantibody subsets for improved diagnostic accuracy. By immobilizing the glycopeptides to AuNPs, a dot blot-based assay was developed to detect autoantibodies in serum. Employing this strategy, in a study involving 20 pancreatic cancer patients and 20 healthy controls, AuNPs containing glycopeptides presenting the GST*A sequence demonstrated the ability to detect autoantibodies in serum. In particular, employing this approach, the detection of autoantibodies outperformed the current pancreatic cancer biomarkers in terms of sensitivity and specificity. Chapter 5 reviews the use of nanoparticle-based delivery of STING agonists for cancer immunotherapy. The mechanism of action of STING, the role of the STING pathway in the cancer cell cycle and the different agonists are described. Subsequently, particular emphasis was placed on the delivery of cyclic dinucleotides, given their role as the natural agonists for STING and their use in Chapter 6 of this thesis. Several types of nanoparticles were described, including inorganic, polymeric, and lipid nanoparticles. The chapter highlights the promising avenue of nanoparticle-based delivery in contrast to the systemic administration of STING agonists as an adjuvant for cancer therapy. Lastly, Chapter 6 explored the use of FDA-approved lipid nanoparticles to deliver an enzymatically resistant cyclic dinucleotide to primary dendritic cells. For the first time, we demonstrated that clinically-approved LNPs could be used for the loading and delivery of cyclic dinucleotides. LNPs exhibited efficient internalization by DCs, releasing the cargo into the cytoplasm, and enhancing STING activation and DC maturation. Additionally, in an in vitro model of tumor-associated human regulatory DCs, these LNPs successfully reprogrammed these cells toward an active state, suggesting their potential for cancer immunotherapy.