{
  "abstract": "Background Developing effective personalized cancer vaccines requires a platform that can rapidly and cost-effectively induce strong CD8+ and CD4+ T cell responses against tumor-specific neoantigens. While plasmid DNA (pDNA) delivered via electroporation (EP) has shown promise in preclinical studies and a Phase I COVID-19 trial, its transition to personalized cancer vaccines is challenged by manufacturing speed and efficiency.Methods To overcome these limitations, we investigated a synthetic approach using linear hairpin (hp)DNA™ delivered by electroporation (EP). This approach leverages the stability of DNA vaccines with the rapid delivery of EP. We assessed immune responses through IFNg ELISpot, spectral flow cytometry, and 10x Genomics single-cell analysis. We then evaluated the anti-tumor effects of the induced T cell responses in three tumor models (MC38, CT26, and E0771), including subcutaneous tumors and MC38 lung metastases.Results We successfully generated and delivered hpDNA-EP vaccines targeting 20 neoantigens in MC38 and CT26 models, and 40 in the E0771 model. We confirmed the immunogenicity of known neoantigens and identified new CD8 + and CD4+ epitopes in E0771 tumors using our prediction pipeline. Benchmarking against pDNA showed that hpDNA-EP at equimolar doses provided significant tumor protection in subcutaneous models, both prophylactically and therapeutically when combined with immune checkpoint inhibitors (ICIs).Deep single-cell analysis of CD8+ T cell responses to the MC38-specific Adpgk neoantigen revealed a Th1 immune profile and the induction of novel T cell receptor (TCR) sequences. Notably, in the lung metastasis model, a mixed-modality vaccination protocol using hpDNA-EP alone or with ICIs significantly reduced metastases, outperforming ICI monotherapy. This marks the first demonstration of a fully synthetic neoantigen cancer vaccine (NCV) based on linear DNA.Conclusions Our findings suggest that hpDNA-EP offers significant advantages for clinical application. The unique TCRs induced by this platform indicate its potential for use in heterologous vaccination protocols to elicit broader and more potent T cell-mediated immune surveillance. This synthetic biology-based NCV is a feasible and promising alternative immunotherapy platform, with the potential to develop more effective and durable cancer immunotherapies, particularly for patients in neoadjuvant settings who currently lack effective therapies to prevent relapse after tumor removal.",
  "authors": [
    {
      "affiliations": [
        "Neomatrix, Roma, Italy"
      ],
      "name": "Fabio Palombo"
    },
    {
      "affiliations": [
        "Neomatrix, Roma, Italy"
      ],
      "name": "Andrea Bianchi"
    },
    {
      "affiliations": [
        "Neomatrix, Roma, Italy"
      ],
      "name": "Ilaria Esposito"
    },
    {
      "affiliations": [
        "Neomatrix, Roma, Italy"
      ],
      "name": "Claudia Tonini"
    },
    {
      "affiliations": [
        "4basebio, Cambridge, Cambridge, UK"
      ],
      "name": "Emily R Young"
    },
    {
      "affiliations": [
        "Neomatrix, Roma, Italy"
      ],
      "name": "Mauro Esposito"
    },
    {
      "affiliations": [
        "4basebio, Cambridge, Cambridge, UK"
      ],
      "name": "Amy Walker"
    },
    {
      "affiliations": [
        "Neomatrix, Roma, Italy"
      ],
      "name": "Luigi Aurisicchio"
    }
  ],
  "title": "1194 Synthetic linear DNA vaccines as a rapid and potent platform for personalized cancer immunotherapy",
  "uid": "769d3993-fa44-5390-b115-c9a7a50bfc63"
}