Background Peptide vaccines are a direct way to initiate an immunogenic response to a defined antigen epitope. However, one of the main disadvantages of anti-tumor peptide vaccines have been their instability, as they are rapidly degraded by enzymatic proteolysis. To overcome this, we developed peptides that would have increased stability against proteolysis by substituting the natural α-amino acid residue with a homologous β-amino acid residue. In another approach we are generating peptides with thioamide substitutions to the backbone amide group to overcome enzymatic proteolysis.
Methods We utilized the ovalbumin antigen model, and developed SIINFEKL peptide analogues with β-substitutions for each of the amino acids. We tested the efficacy of the modified peptides against the native peptide in vitro for their immunogenic properties. The in vitro studies tested the peptides for their capability to activate antigen specific CD8 T cells and to identify the T cell phenotype resulting from this stimulation. Specifically, splenocytes from OT-1 mice were incubated with native/modified peptide and then analyzed via flow cytometry.
Results We report that each of the β-substitution on the SIINFEKL peptide were capable of activating antigen specific CD8 T cells in the in vitro assay, which is demonstrated by the increased expression of 4–1BB, IFN-γ, TNF-α and PD-1. We also report that after stimulation with native/modified peptides the antigen specific CD8 T cells expressed cytokines such as IL2, IL4, IL10 and IL17a. Interestingly, the β-substitution at the fifth amino acid (F - Phenylalanine) activated the T cells to the same level as the native peptide but resulted in lower PD-1 expression. We also report that this modified epitope was more potent than the other modifications as it was able to generate an immunogenic activity at a low concentration of 100 pg/ml whereas other substitutions failed to do so.
Conclusions Modified (β-substituted) analogues of the antigenic peptide have been shown to improve the stability of the peptides against enzymatic proteolysis.1 We demonstrated that these modifications can also improve their immunogenic potential by resulting in T cells with a favorable phenotype of lower checkpoint molecule expression while maintaining activation. Ongoing studies are evaluating the thioamide substitutions to SIINFEKL and the capability of these modified peptide analogues to elicit an immunogenic response in vivo.
Webb AI, Dunstone MA, Williamson NA, Price JD, de Kauwe A, Chen W, Oakley A, Perlmutter P, McCluskey J, Aguilar MI, Rossjohn J, Purcell AW. T cell determinants incorporating beta-amino acid residues are protease resistant and remain immunogenic in vivo. J Immunol. 2005 Sep 15;175(6):3810–8.
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