Synthesis of dual adjuvanted peptide vaccines for cancer immunotherapy
Authors:   , Mathew Juby 
Published: January 2025

<p dir="ltr">In 2013, editors of the prestigious journal <i>Science</i>, hailed Cancer Immunotherapy as the “breakthrough of the year.” Cancer Immunotherapy uses the body’s own immune system to attack cancer cells. To date, the most successful cancer immunotherapies work by blocking tumour-induced immunosuppressive signals whereas those based on boosting immune responses (i.e. vaccination) have been less successful. Therefore there is still a need to develop better vaccines that induce stronger and more sustained immune responses. By inducing potent immunity, prophylactic vaccination has tremendously helped in controlling mortality caused by infectious diseases through, mainly, the induction of antibody responses. In contrast, therapeutic vaccination that generates potent cytotoxic T lymphocyte (CTL) responses can be used to treat cancer. Peptide-based vaccines are a type of subunit vaccine that incorporate the most basic antigenic components (avoiding off-target effects of other vaccine vectors). Due to their well-defined nature that aids in manufacture, safety considerations and redesign, peptide-based vaccines has piqued the interest of vaccine developers. Despite this and although highly versatile, a major limitation with peptide-based vaccines is their lack of immunogenicity. The addition of a vaccine adjuvant and/or formulation are common strategies often employed to overcome this limitation. Rational vaccine design can be adapted from natural immunity, where the immune system responds to pathogens which are antigens co-delivered with a wide variety of inflammatory signals foreign to the host. With this in mind, covalent conjugation of the antigen and adjuvant can improve vaccine efficacy as it aims to co-localise both the antigen and immune stimulant to the same cell mimicking this aspect of natural infection.</p><p dir="ltr">In vaccine development, Toll-like receptor (TLR) agonists such as unmethylated CpG (5′ – C-phosphate-G – 3′) oligonucleotide and resiquimod, that directly activate antigen presenting cells (APCs) are commonly used vaccine adjuvants. A different approach utilizes glycolipid molecules, such as α-galactosylceramide (α-GalCer), to activate invariant natural killer T (iNKT) cells. Once activated, these cellular adjuvants (i.e. the iNKT cells) activate APCs including dendritic cells (DCs) through the provision of CD40L and cytokines. This alternative pathway can be considered similar to the classical CD4-T cell-mediated DC-licensing pathway. Licensed or activated DCs (via either pathway) can drive the expansion of naive CD8+ T cells into CTLs specific to the antigen being co-presented. Although the end result is similar, there are reported qualitative differences in the T cells produced. Thus, the combination of TLR ligands and iNKT cell agonists may be used to overcome the modest results obtained from their individual use by fine-tuning DC maturation and synergistic CTL priming.</p><p dir="ltr">The aim of this thesis was to investigate if the covalent attachment of two adjuvants (i.e. TLR and NKT cell ligands) to an antigenic peptide would improve vaccine efficacy. To this end, we successfully synthesised conjugates using three different chemical designs: -</p><ol><li>Triconjugate I – branched with branching point at the peptide N-terminus (Chapter 3).</li><li>Triconjugate II – linear with the peptide located centrally and adjuvants at either end (Chapter 3).</li><li>Triconjugate III – branched using a glutamic acid derivative-based trivalent core (Chapter 4).</li></ol><p dir="ltr">Because TLR or NKT cell stimulation is known to be toxic in certain situations and therefore the combination may be even more toxic, a preliminary aim of this thesis was to investigate if the hepatocyte damage, as seen by altered histology and a transient rise in levels of transaminases in mice, associated with administration of α-GalCer can be modulated by utilising structural analogues of this prototypical NKT cell ligand such as derivatives with shortened acyl chain length. A series of glycolipid-peptide vaccines were synthesised where the acyl chain has been reduced progressively from C26 to C0 (Chapter 2). Data from the preliminary biological studies using a model antigen suggest shorter acyl chain analogues can achieve NKT cell activation with minimal liver toxicity. Thus, shortening the fatty acyl chain may be a favourable strategy to explore further.</p><p dir="ltr">Due to outside events towards the end of this thesis, biological studies to assess the therapeutic potential of these conjugates was not completed, however, they are currently ongoing.</p>