{
  "abstract": "Background Functional loss of tumor suppressor genes encoded by the CDKN2A locus (p16INK4A and p14ARF) contributes to tumorigenesis. However, p14ARF is up-regulated when its downstream effectors are inactivated, and is overexpressed in human tumors including most tumors with p53 mutations, 1 making it a candidate tumor-associated target. p14ARF, a nuclear protein, is inaccessible to CAR-T cell or antibody therapeutics but can be targeted by TCR-based therapies recognizing target-derived peptides presented on HLA molecules on the cell surface. Here, we identify and characterize TCRs specific for a p14ARF-derived peptide displayed on HLA-A*02:01. TCR-transduced T cells (TCR-T) expressing this TCR mediated in vivo efficacy against p14ARF+ tumors.Methods A well-presented epitope from p14ARF was identified and healthy donor repertoires screened for high affinity p14ARF-specific TCRs. TRA/TRB genes were cloned and assembled as codon-optimized lentiviral vectors to generate TCR-T cells. TCR-T cells were evaluated for antigen-specific activation, cytokine production, and in vitro elimination of p14ARF-expressing targets, and TCR specificity was assessed by X-scanning.2 TCR-T cell in vivo efficacy was characterized in p14ARF-expressing tumor xenograft models. Safety of targeting p14ARF in vivo was assessed using a murine ARF-GFP model in which the endogenous p14ARF coding sequence is replaced by GFP.3 GFP-reactive JEDI transgenic T cells were adoptively transferred into ARF-GFP mice to monitor the impact of eliminating p14ARF-expressing cells in normal tissues.Results Seven p14ARF-specific TCRs were identified and TCR-T cells expressing these TCRs were evaluated in vitro and in vivo. Two of these TCRs killed HeLa cells (endogenously expressing p14ARF) at a low effector to target ratio, but not HeLa cells made deficient for p14ARF. X-scan analysis of these TCRs identified four potential off-target peptides; however, these epitopes were not efficiently processed or presented under physiological conditions. To study potential on-target off-tumor effects of targeting p14ARF-expressing cells with CD8+ T cells in vivo, GFP-specific JEDI T cells were transferred into ARF-GFP mice. No impact was observed on blood cell counts, blood chemistry, or body weight. Notably, p14ARF-targeted TCR-T cells inhibited growth of established p14ARF expressing tumors in vivo.Conclusions A human TCR specific for a p14ARF-derived peptide well-presented by HLA-A*02:01, yielded a TCR T-cell therapy that selectively and potently lysed p14ARF-expressing cancer cells in vitro and inhibited tumor growth of p14ARF+ tumors in vivo. Our studies suggest eliminating p14ARF+ cells in normal tissues is well tolerated in vivo. Thus, p14ARF-specific TCR-T cells emerge as a promising novel therapy for various cancers overexpressing this tumor suppressor.References Inoue K, Fry EA. Aberrant expression of p14(ARF) in human cancers: a new biomarker? Tumor Microenviron 2018;1:37-44. doi:10.4103/tme.tme_24_17Harper J, et al. An approved in vitro approach to preclinical safety and efficacy evaluation of engineered T cell receptor anti-CD3 bispecific (ImmTAC) molecules. PLoS One 2018;13:e0205491. doi:10.1371/journal.pone.0205491Sherr CJ. An Arf(GFP/GFP) reporter mouse reveals that the Arf tumor suppressor monitors latent oncogenic signals in vivo. Cell Cycle 2004;3:239-240. doi:10.4161/cc.3.3.674",
  "authors": [
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Thomas M Schmitt"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Kelsey Furiya"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Cheryl Black"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Angie Vazquez"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Menna Hailemariam"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Daniel Paushter"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Lam Trieu"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Jennifer Lam"
    },
    {
      "affiliations": [
        "Cullinan Therapeutics, Cambridge, MA, USA"
      ],
      "name": "Kavya Rakhra"
    },
    {
      "affiliations": [
        "Cullinan Therapeutics, Cambridge, MA, USA"
      ],
      "name": "Karsten Sauer"
    },
    {
      "affiliations": [
        "Cullinan Therapeutics, Cambridge, MA, USA"
      ],
      "name": "Patrick A Baeuerle"
    },
    {
      "affiliations": [
        "Cullinan Therapeutics, Cambridge, MA, USA"
      ],
      "name": "Jennifer S Michaelson"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Philip D Greenberg"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Aude G Chapuis"
    }
  ],
  "title": "344 Safety and efficacy of targeting the p14ARF tumor suppressor using TCR T-cell therapy",
  "uid": "402b9506-8082-5123-a2d4-a64c19963568"
}