{
  "abstract": "Background Peptide-based vaccines offer a targeted strategy for eliciting CD8+ T cell responses against tumor antigens but are limited by rapid degradation due to enzymatic proteolysis. To address this, we designed peptide analogues containing thioamide substitutions—replacing backbone amides while retaining native side chains—to enhance proteolytic stability without altering side chain interactions necessary for MHC I binding and TCR recognition. While most chemical modifications hinder peptide-MHC (pMHC) formation or T cell recognition, thioamides provide a subtle backbone modification that could preserve or even improve immunogenicity. We hypothesized that thio-substituted peptides (‘thio-antigens’) would remain immunogenic and induce antigen-specific CD8+ T cell responses with potentially favorable phenotypes.Methods Using the ovalbumin-derived SIINFEKL model epitope, we synthesized a panel of peptides with single, double, and triple thioamide substitutions at different positions. These analogues were tested for their capacity to activate OT-I CD8+ T cells in vitro and to elicit immunogenic responses in vivo in C57BL/6 mice. T cell phenotype was assessed by expression of activation, memory and exhaustion related markers by flow cytometry. Antigen-specific CD8+ T cells were quantified via tetramer staining, and tumor control was evaluated in a syngeneic tumor model where thio-antigens were delivered with TLR agonists after adoptive transfer of OT-I splenocytes.Results Most thio-antigens retained the ability to activate CD8+ T cells in vitro, with five of seven single-substituted peptides inducing equal or greater expression of 4-1BB on OT-I cells after stimulation compared to native SIINFEKL. Substitutions at Ile2 and Asn4 resulted in reduced activation, while others preserved or enhanced T cell responses. Several thio-antigens induced CD8+ T cells with altered PD-1 expression, suggesting a distinct activation phenotype. In vivo, thio-antigen vaccination generated antigen-specific effector-memory CD8+ T cells, some with enhanced 4-1BB and PD-1 expression. Notably, a thio-substitution at Ser1 resulted in enlarged spleen sizes, indicating possible systemic effects. CD8 T cells elicited or activated by thio-antigens retained specificity for the native SIINFEKL epitope, as demonstrated by cross-reactivity with the native epitope in ELISpot assays. Selected thio-antigens conferred tumor control comparable to the native peptide.Conclusions Thio-antigens are a promising strategy to enhance the stability and modulate the immunogenicity of peptide vaccines. Our findings support further development of thio-antigens as candidates for cancer immunotherapy, potentially yielding more durable and functionally distinct CD8+ T cell responses compared to conventional peptide vaccines.",
  "authors": [
    {
      "affiliations": [
        "University of Wisconsin Madison, Madison, WI, USA"
      ],
      "name": "Ichwaku Rastogi"
    },
    {
      "affiliations": [
        "University of Wisconsin Madison, Madison, WI, USA"
      ],
      "name": "John A Mannone"
    },
    {
      "affiliations": [
        "University of Wisconsin Madison, Madison, WI, USA"
      ],
      "name": "Akash M Sarkar"
    },
    {
      "affiliations": [
        "Indian Institute of Science, Bangalore, Karnataka, India"
      ],
      "name": "Nishant Raj"
    },
    {
      "affiliations": [
        "Indian Institute of Science, Bangalore, Karnataka, India"
      ],
      "name": "Jayanta Chatterjee"
    },
    {
      "affiliations": [
        "University of Wisconsin Madison, Madison, WI, USA"
      ],
      "name": "Samuel H Gellman"
    },
    {
      "affiliations": [
        "University of Wisconsin Madison, Madison, WI, USA"
      ],
      "name": "Douglas G McNeel"
    }
  ],
  "title": "935 Thioamide-substituted peptide analogues retain antigenicity and modulate CD8+ T cell activation in anti-tumor vaccine models",
  "uid": "e6dce818-83e4-58e6-9b05-892711faf5f9"
}