Investigation of antigen loading in synthetic peptide vaccines for cancer immunotherapy
Vaccines that generate cytotoxic T lymphocyte (CTL) responses can be used to treat non communicable diseases such as cancer or chronic infection. However, there are few established vaccine platforms that generate responses of sufficient quality and magnitude that have translated into routine clinical application.
One therapeutic approach utilises vaccines based on peptide antigens. Peptide based vaccines are fully synthetic and therefore have considerable manufacturing and design advantages compared to other more complicated vaccine concepts, which may require biological processes such as fermentation and/or protein expression or the isolation and further manipulation of host cells. However, a major consideration in the design of a peptide vaccine is the low immunogenicity of peptide antigens. As such, peptide antigens must be delivered in specific ways and/or accompanied by an immune stimulant (i.e. an adjuvant compound).
Adjuvant compounds often work by triggering pattern recognition receptors such as the toll-like receptors (TLRs). There are various TLR adjuvants in clinical development, one of interest is resiquimod. Resiquimod has shown to be a promising cancer immunotherapeutic adjuvant and has proven ability to secrete cytokines, upregulate costimulatory molecule expression, and enhance antigen presentation to T cells resulting in the induction of antigen specific CD8+ T cell responses. Another mechanism involves the activation and proliferation of antigen specific CD4+ T-helper cells to support antibody production and/or the proliferation and differentiation of naive CD8+ T cells into CTLs. Additionally to this, ‘alternative help’ can be supplied by ancillary cells such as innate-like T cells. These cells contain a T-cell receptor (TCR), but unlike conventional T cells which take days to respond to stimuli, they can respond in minutes or hours. Invariant natural killer T (iNKT) cells are a well-studied innate-like T cell population and in contrast to conventional T cells that respond to peptide antigens presented by major histocompatibility complex (MHC) molecules, iNKTs respond to lipid antigens presented by the MHC class I like molecule CD1d. Engagement of the iNKT TCR with lipid antigens (such as α-galactosylceramide, α-GalCer) presented in the context of CD1d, results in the activation of the iNKT cells and a positive feedback loop of dendritic cell (DC) activation through provision of CD40 ligand (CD40L) which ultimately enhances T cell responses.
In the case of peptide vaccines, chemical conjugation of the adjuvant and peptide antigen for either TLR or iNKT adjuvanted vaccines has been shown to further enhance immunogenicity compared to admixed controls. Most chemical conjugation approaches are limited to an equal molar ratio of adjuvant to antigen, whereas most vaccine formulations consist of higher molar amounts of peptide antigens compared to adjuvant. We therefore hypothesised that chemical conjugates containing increased amounts of peptide antigen would be more immunogenic and efficacious than those with lower peptide antigen.
To investigate this hypothesis, a series of dendron vaccines were synthesised that contained one to eight copies of peptide antigen to adjuvant molecules. During the course of this research various adjuvants (i.e. α-GalCer and α-galactosylphytosphingosphine (α-GalPhs), and a derivatised form of the TLR 7/8 agonist resiquimod), protease-cleavable linkers, and conjugation chemistries were investigated in the context of a PEE-G dendron core. Surprisingly, installation of the cyclooctyne moiety for use in strain-promoted alkyne-azide cycloaddition (SPAAC) conjugation methodology proved difficult and was ultimately replaced with oxime ligation.
Data generated on the vaccines synthesised confirmed conjugation indeed improves immunogenicity, however for admixes or conjugates there was little observable advantage gained by increasing the amount of antigen. Although further investigations will be required to completely disprove the hypothesis, the compounds synthesised and tested have opened up new areas of chemical and immunological investigation. In particular, the multigenerational branched conjugate design could generate strong T cell responses without eliciting potent innate immune stimulation which could be used to lower the impact of therapeutic vaccination.