{
  "abstract": "Background Chimeric antigen receptor (CAR) T therapy has seen limited success in patients with solid tumors, likely due to a combination of physical barriers, metabolic challenges, and T cell exhaustion. 1 As such, this powerful therapy has remained inaccessible to a large cohort of patients. Rhabdomyosarcoma (RMS), the most common pediatric soft-tissue sarcoma, has seen few advances in therapies over the last decades.2 These tumors exhibit low T cell infiltrate, making them resistant to checkpoint immunotherapy. CART therapy offers the opportunity to direct sarcoma-specific T cells into the tumor microenvironment (TME), overcoming this lack of anti-tumor response. However, as in other solid tumors, CART therapy is showing limited efficacy in sarcoma clinical trials.3 Here we use an immunocompetent preclinical model of RMS to assess the TME and the role of inhibitory receptors (IR) following CART therapy.Methods The murine RMS cell line M39M 4 is injected intramuscularly into C57BL6 mice. Following preconditioning with cyclophosphamide, murine CART cells specific for the sarcoma antigen B7-H35 are injected intravenously. Tumor growth is followed and the TME evaluated at predetermined time points. To monitor killing capacity in vitro, CARTs are mixed with M39M cells and monitored using the xCELLigence RTCA eSight (Agilent). Multiple murine models and CRISPR/Cas9 technology were used to assess the role of IRs PD1 and LAG3 on CART function.Results Murine B7-H3 CARTs efficiently infiltrate M39M tumors, where they are the majority of intra-tumoral CD8+ T cells by day 7 post-injection. These CARTs exhibit high expression of IRs, including PD1, LAG3, and Tim3. Despite high CART numbers isolated from the TME, there is no significant impact of CARTs alone on tumor growth. Preliminary analysis of the TME reveals an influx of myeloid-derived suppressor cells as well as increased expression of PDL1 by immune and tumor cells. In vitro, M39M expresses little PDL1 at baseline, whereas CART exposure leads to high PDL1 expression. In this setting, CART cells without PD1 demonstrate improved killing at low E:T ratios, with a particular advantage to PD1/LAG3 dual deficient CARTs.Conclusions While B7-H3 CARTs can efficiently target murine RMS, they do not limit tumor growth. Analysis of the TME suggests that CART infiltration creates a more immunosuppressive TME with high PDL1 expression, and in vitro data support a role for IR blockade in improving CART therapy against RMS. Ongoing studies are evaluating additional mechanisms of resistance in the TME to design effective combination strategies to improve CART therapy for patients with sarcoma.References Ramakrishna S, Barsan V, Mackall C. Prospects and challenges for use of CAR T cell therapies in solid tumors. Expert Opin Biol Ther. 2020 May;20(5):503–516. PMID: 32125191Zarrabi A, Perrin D, Kavoosi M, Sommer M, Sezen S, Mehrbod P, Bhushan B, Machaj F, Rosik J, Kawalec P, Afifi S, Bolandi SM, Koleini P, Taheri M, Madrakian T, Łos MJ, Lindsey B, Cakir N, Zarepour A, Hushmandi K, Fallah A, Koc B, Khosravi A, Ahmadi M, Logue S, Orive G, Pecic S, Gordon JW, Ghavami S. Rhabdomyosarcoma: current therapy, challenges, and future approaches to treatment strategies. Cancers (Basel). 2023 Nov 2;15(21). PMCID: PMC10650215Pinto NR, Albert CM, Taylor M, Wilson A, Rawlings-Rhea S, Huang W, Seidel K, Narayanaswany P, Wu V, Brown C, Vitanza NA, Orentas R, Gardner RA, Jensen MC, Park JR. STRIVE-02: A first-in-human phase 1 trial of systemic B7H3 CAR T cells for children and young adults with relapsed/refractory solid tumors. JCO. 2022 Jun 1;40(16_suppl):10011–10011.Meadors JL, Cui Y, Chen Q-R, Song YK, Khan J, Merlino G, Tsokos M, Orentas RJ, Mackall CL. Murine rhabdomyosarcoma is immunogenic and responsive to T-cell-based immunotherapy. Pediatr Blood Cancer. 2011 Dec 1;57(6):921–929. PMCID: PMC7401311Du H, Hirabayashi K, Ahn S, Kren NP, Montgomery SA, Wang X, Tiruthani K, Mirlekar B, Michaud D, Greene K, Herrera SG, Xu Y, Sun C, Chen Y, Ma X, Ferrone CR, Pylayeva-Gupta Y, Yeh JJ, Liu R, Savoldo B, Ferrone S, Dotti G. Antitumor responses in the absence of toxicity in solid tumors by targeting B7-H3 via chimeric antigen receptor T cells. Cancer Cell. 2019 Feb 11;35(2):221-237.e8. PMCID: PMC6645919",
  "authors": [
    {
      "affiliations": [
        "University of Pittsburgh, Pittsburgh, PA, USA"
      ],
      "name": "Erica Braverman"
    },
    {
      "affiliations": [
        "University of Pittsburgh, Pittsburgh, PA, USA",
        "University of Pittsburgh School of Medicine, Pittsburgh, PA, USA"
      ],
      "name": "Sebastian Hymson"
    },
    {
      "affiliations": [
        "University of Pittsburgh, Pittsburgh, PA, USA"
      ],
      "name": "Alexa Jones"
    },
    {
      "affiliations": [
        "University of Pittsburgh, Pittsburgh, PA, USA"
      ],
      "name": "Nicole Mihalik"
    },
    {
      "affiliations": [
        "University of Pittsburgh, Pittsburgh, PA, USA"
      ],
      "name": "Andrew Baessler"
    },
    {
      "affiliations": [
        "University of Pittsburgh, Pittsburgh, PA, USA"
      ],
      "name": "Creg Workman"
    },
    {
      "affiliations": [
        "University of Pittsburgh, Pittsburgh, PA, USA"
      ],
      "name": "Dario AA Vignali"
    }
  ],
  "title": "203 Overcoming CART therapy resistance in an immunocompetent model of rhabdomyosarcoma",
  "uid": "f9e20d95-34d9-5131-8113-7d438654b552"
}