Development of cancer nanovaccines based on glyconeoantigens

• Autor(a) principal: Freitas, Rui Filipe Neves
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/30109
• topic: Glycosylation; Cancer nanovaccines; Neoantigens

Cancer vaccines exploiting cancer neoantigens have been proven capable of eliciting powerful immune responses against cancer cells with limited offtarget effects, constituting the next cornerstone in cancer treatment. Cancer vaccines are also capable of inducing immunological memory against recurrences, which is key for cancer management. The most promising epitopes remain peptides derived from genomic mutations. However, many solid tumours present a low mutational burden, hampering the generalization of these approaches and demanding the exploitation of other classes of biomolecules. Alterations in protein glycosylation translated by the expression of short-chain O-glycans at the surface of cancer cells offers tremendous potential towards this objective. Despite their cancer-associated nature and relevant role in cancer development, these glycans also contribute to immunosuppression, frustrating attempts to develop glycan-based vaccines. Envisaging this goal, this work describes a single-pot chemoenzymatic method for synthesizing glycoepitopes derived from cancer-associated Mucin16 (MUC16) expressing different classes of neutral and sialylated short-chain O-glycans in mimicry of cancer cells. In parallel, MUC16-Tn glycopeptides wereconjugated with immunogenic protein key lymphocyte hemocyanin (KLH), foreseeing the development of conventional glycan-protein vaccine constructs. The second part of the work exploits the potential of nanoconstructs for improved vaccine formulations. Briefly, PLGA nanoparticles were functionalized or adsorbed with MUC16-Tn and characterized by TEM and 1H NMR. Nanoparticles showed a spherical and homogenous nature, low polydispersion index, dimensions bellow 200nm and high stability at physiological pH. Toxicity thresholds and internalization capacity by antigen presenting cells was determined using macrophages. The third part of the work concerned the development of a cell model expressing the Tn antigen which was used to infer the functional implications of this antigen, with emphasis on immune evasion. Briefly, the genome of T24 urothelial cells was edited through CRISPR/Cas9 technology to produce a stable C1GALT1 knock-out. This cell line expressed significantly high levels of the Tn antigen at the cell surface and completely obliterated the expression of extended glycoforms, as translated by flow cytometry, immunofluorescence microscopy and glycomics analysis by mass spectrometry. Moreover, glycoproteomics studies confirmed MUC16-Tn expression, supporting the utility of the model in the context of the developed vaccine constructs. Interestingly, Tn antigen expression did not impact on cell proliferation, migration and invasion as well as in the expression of important molecules for cancer immune escape. Nevertheless, co-culture assays with dendritic cells suggested that Tn expressing cancer cells may negatively influence their differentiation and downregulate relevant molecules involved in antigen presentation. In summary, a rationale has been created to support the development and improvement of glycan-based vaccines for cancer foreseeing in vivo studies. Given the pancarcinomic nature of these glycoepitopes, the generalization of more efficient vaccines to different cell models may be envisaged.