Hyaluronan as an immunological adjuvant in protein- and peptide-based cancer vaccines: applications in HER2/neu-expressing breast cancer and insights into mechanisms of action

Cancer vaccines represent one of the most promising strategies in modern immunotherapy, but their translation to oncology has achieved limited clinical success. Protein- and peptide-based anticancer vaccines offer advantages in terms of safety, specificity, and manufacturing flexibility, but their efficacy depends on adjuvants capable of inducing robust and durable immune responses against weakly immunogenic or self-derived tumor antigens. Within this framework, the laboratory hosting this thesis patented a vaccine platform based on 200 kDa hyaluronic acid (HA) chemically conjugated to protein or peptide antigens. Studies using model antigens and antiviral applications demonstrated that HA-based bioconjugates induce strong and long-lasting antigen-specific humoral and cellular immune responses while maintaining a favorable safety profile. Studies with HA conjugated to ovalbumin (OVA) suggested that HA-bioconjugates can drain from the injection site to the draining lymph node, where they promote antigen presentation and immune priming. Based on its physicochemical and immunological properties, HA is hypothesized to act as an endogenous danger-associated molecular pattern (DAMP), potentially involving TLR2- and TLR4-mediated signaling pathways. Understanding the mechanism of action of this platform is essential to optimize vaccine design and support its clinical translation. The first aim of this thesis was to investigate the immunological activity and mechanism of action of HA-based vaccines using OVA as a model antigen. HA-OVA bioconjugates were evaluated in C57BL/6 wild-type, TLR2-knockout, and TLR4-knockout mice to assess the contribution of innate immune signaling pathways to the adjuvant activity of HA. Vaccination with HA-OVA induced strong antigen-specific humoral immunity, characterized by increased anti-OVA antibody production compared with control formulations and durable humoral memory supported by the persistence of OVA-specific long-lived plasma cells up to one year after immunization. Analyses suggested a differential contribution of TLR2 and TLR4 signaling pathways to the immune response elicited by HA conjugation. TLR2 appeared to contribute to the establishment of long-lived humoral immunity, whereas TLR4 signaling was associated with antigen-specific cytotoxic responses in in vivo T-cell killing assays. Translational studies focused on the identification of HER2-derived extracellular peptides for the development of multipeptide vaccination strategies against HER2-positive breast cancer, an aggressive tumor subtype associated with poor prognosis and high risk of recurrence. Building on unpublished data demonstrating the efficacy of HA-HER2 protein-based vaccination, this work aimed to identify shared HER2-derived peptides with broad predicted HLA coverage and favorable immunogenic potential, suitable for off-the-shelf vaccine approaches. Preliminary screening using individual peptide formulations allowed the identification of four candidate peptides for multipeptide vaccine development. Ongoing studies are evaluating pooled peptide formulations in combination with clinically relevant adjuvants employed in cancer vaccination, including AS04 and Montanide ISA 51. Proof-of-concept data with an HA-conjugated peptide formulation support the potential applicability and superiority of the HA platform in peptide-based anticancer vaccination strategies. Overall, this work supports HA conjugation as a promising strategy to enhance antigen-specific immunity and highlights the potential of HA-based platforms for the development of safe and effective protein- and peptide-based anticancer vaccines.