{
  "abstract": "Background Chimeric antigen receptor (CAR) T cell therapy in solid tumors has been limited by challenges such as antigen heterogeneity. EPHA2, a receptor tyrosine kinase, is a stress antigen and it is frequently overexpressed in multiple malignancies, including osteosarcoma (OS) and non-small cell lung cancer (NSCLC), where it is associated with aggressive tumor biology and metastatic progression. 1–5 As a proof of concept for targeting EPHA2 in both adult and pediatric cancers, our project aims to preclinically validate EPHA2-directed CAR T cells using aggressive in vivo models of NSCLC and OS, with the goal of enhancing tumor clearance and immune activation.Methods 24 fully human EPHA2-specific antibodies were selected based on strong binding to soluble and cell-surface EPHA2 (EC50 < 25 nM by ELISA). Second-generation CAR T cells were engineered and screened in vitro using luciferase-based cytotoxicity assays and live-cell imaging against high EPHA2-expressing A549, H1993 (NSCLC) and 143B, SAOS2 (OS) cell lines. The top 3 candidates were evaluated in vivo in metastatic 143B and A549 xenograft models in MHC DKO mice. Tumor burden by bioluminescence imaging and time to disease progression were assessed and compared across treatment groups.Results In cytotoxicity assays, 75% growth inhibition in A549 and 90% in 143B was seen with the lead CAR T construct (EP10) compared to 41% and 53% with a contemporaneous construct, 4H5, respectively (1:8 Effector:Target ratio). Significant reduction in tumor burden with EP10 was also observed in A549 (p<0.0001, 2-way ANOVA) and 143B xenografts (p<0.005) compared to 4H5 and a non-targeting control (p<0.0001 in A549 and 143B). Significant progression free survival was also observed in EP10 treated A549 model (vs 4H5, p<0.0005, median survival (MS) 115 days; vs control, p<0.0001, MS 105 days); notably, 75% of the mice treated with EP10 survived until day 200 when the study concluded, and median survival was not reached. In the aggressive 143B model EP10 also conferred survival benefit (MS 54 days, vs 4H5, p<0.01, MS 49 days; vs control, p<0.005, MS 44 days).Conclusions We describe initial development of a novel EPHA2-targeted CAR T cells with superior cytotoxicity, tumor control, and survival benefit over a contemporaneous product in both OS and NSCLC models. Construct optimization with immunomodulatory cytokine armoring is ongoing and expected to further enhance its anti-tumor efficacy. Results from these studies will provide preclinical foundation for development of an investigator-initiated clinical trials in relapsed or metastatic cancer patients.Ethics Approval For PDX related studies, patient consent was obtained through IRB #12-245 and/or #06-107. Animal studies were conducted under IACUC-approved protocol #16-08-011 and #00-05-065.References Toracchio L, Carrabotta M, Mancarella C, Morrione A, Scotlandi K. EphA2 in Cancer: Molecular complexity and therapeutic opportunities . Int J Mol Sci. 2024;25(22).Wilson K, Shiuan E, Brantley-Sieders DM. Oncogenic functions and therapeutic targeting of EphA2 in cancer. Oncogene. 2021;40(14):2483-95.Kinch MS, Moore MB, Harpole DH, Jr. Predictive value of the EphA2 receptor tyrosine kinase in lung cancer recurrence and survival. Clin Cancer Res. 2003;9(2):613-8.Giordano G, Tucciarello C, Merlini A, Cutrupi S, Pignochino Y. Targeting the EphA2 pathway: could it be the way for bone sarcomas? Cell Commun Signal. 2024;22(1):433.Diakogiannaki I, D’Amore VM, Affinito A, Donati G, Cinquegrana E, Quintavalle C, et al. Targeting glioblastoma stem cells via EphA2: structural insights into the RNA aptamer A40s for precision therapy. J Chem Inf Model. 2025;65(11):5635-48.",
  "authors": [
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Ali Cihan"
    },
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Daoqi You"
    },
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Armaan Siddiquee"
    },
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Kristina Guillan"
    },
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Tamar Feinberg"
    },
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Erin Burns"
    },
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Jasmine Um"
    },
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Joan Rou-En Choo"
    },
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Andrew L Kung"
    },
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Anthony F Daniyan"
    },
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Filemon Dela Cruz"
    }
  ],
  "title": "212 Development of EPHA2 CAR T cell therapy for solid tumors",
  "uid": "9fecd1a5-a8c8-5493-892d-4c64555054b9"
}