{
  "abstract": "Background Ovarian cancer is one of the deadliest gynecological cancers in the US. Patients with advanced disease have a 5-year survival rate of 31%, underscoring the need for innovative treatment strategies. Engineering T cells to target tumor-specific proteins could enhance treatment efficacy while minimizing toxicity to healthy tissues. Mesothelin (Msln) is minimally expressed in healthy tissues, overexpressed in ovarian cancer cells and contributes to malignancy, making it an excellent immunotherapy target. In a mouse model, T cells engineered to express a Msln-targeting T cell receptor (TCR Msln) delayed tumor growth, but inhibitory signaling in the tumor microenvironment diminished T cell function and resulted in disease progression. We hypothesized combining engineered T cells and checkpoint blockade would increase treatment efficacy by enhancing both engineered and endogenous immune responses.Methods Eight-week-old female C57BL/6J mice were intraperitoneally inoculated with ID8 VEGF tumors. Six to eight weeks later, mice were lymphodepleted with cyclophosphamide and received TCRMsln-engineered T cells and Msln peptide-pulsed irradiated splenocytes as a vaccine. To support T cell persistence, IL-2 was injected subcutaneously for 10 days post-infusion. Mice were treated with isotype, anti-PD1, anti-Lag-3, or anti-Tim-3 checkpoint antibodies, alone or in combination, starting the day of T cell infusion and re-administered every three days for 21 days. On Day 21, tumors were harvested and tumor-infiltrating lymphocytes (TILs) were sorted for single-cell analysis. RNAseq and CITEseq assay files were processed with Cell Ranger, and the output matrix files were analyzed in R.Results Combining TCR Msln T cells with PD-1, Tim-3, and Lag-3 triple checkpoint blockade led to significantly improved TCRMsln T cell function and overall animal survival. Single-cell RNA sequencing revealed distinct gene expression changes in tumor infiltrating lymphocytes after treatment. Given the critical role of CD4 T cells in cancer immunity, we examined the impact of each treatment on CD4 T cell differentiation. CD4 T cells in control tumors expressed stem-like signatures. After treatment with TCRMsln T cells, tumor-infiltrating CD4 T cells expressed genes associated with TH2 differentiation. Checkpoint blockade alone led to signatures associated with T cell activation but no change in tumor control. CD4 T cells exposed to the TCRMsln T cells and triple checkpoint combination treatment increased expression of pro-inflammatory cytolytic gene signatures.Conclusions These results suggest combination immunotherapy may dramatically alter endogenous immune responses, which could potentially be leveraged to rationally improve anti-tumor efficacy and ovarian cancer patient outcomes.",
  "authors": [
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Asmaa O Mohamed"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Kristin G Anderson"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Gabriel F Alencar"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Madison G Burnett"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Yapeng Su"
    },
    {
      "affiliations": [
        "University Hospital of Lausanne, Lausanne, Lausanne, Switzerland"
      ],
      "name": "Raphael Gottardo"
    },
    {
      "affiliations": [
        "University of Virginia, Charlottesville, VA, USA"
      ],
      "name": "Stefan Bekiranov"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Philip D Greenberg"
    }
  ],
  "title": "339 Engineered T cell therapy combined with PD-1, Lag-3 and Tim-3 checkpoint inhibition promotes pro-inflammatory CD4 T cell differentiation in an ovarian cancer mouse model",
  "uid": "8920af40-7728-552b-8b68-61cb4fb96dad"
}