BackgroundThe 5-year disease-free survival for children and young adults with metastatic sarcoma at diagnosis or recurrent disease after front-line therapy is 20–30%.1 2 Cellular immunotherapy using chimeric antigen receptor (CAR) T cells has shown dramatic benefits in leukemia, but only limited success in solid tumors.3 4 One limitation of CAR T cell therapy has been poor trafficking into solid tumors.5–7 Chemokines are small, secreted, cytokine-like molecules that mediate lymphocyte homing and migration.8 In this study, we discovered that both osteosarcoma (OS) and rhabdomyosarcoma (RMS) cells significantly increase expression of the chemokine IL-8 after clinically achievable doses of radiation, but not at rest. Given that CAR T cells do not express the receptor for IL-8, we created a construct with an IL-8 receptor (CXCR2) and a B7H3 CAR in T cells to improve CAR T homing and to create an effective new immunotherapy for patients with sarcoma.MethodsMultiple OS and RMS cell lines were irradiated at 10 Gy and IL-8 was measured by ELISA. We created retroviral constructs, B7H3 CAR-T2a-CXCR2 and B7H3 CAR. Peripheral blood T lymphocytes were stimulated with IL-2 and anti-CD3/28 antibodies for 48 hours prior to transduction with the retroviral vectors. Surface expression of the scFv (by L protein) and CXCR2 (mAb) were assessed using flow cytometry. In vitro cytotoxicity assays using sarcoma tumor spheroids were conducted using Incucyte. INF-γ and IL-2 production were measured by ELISA. NSG mice injected orthotopically with an IL-8 overexpressing RMS cell line were treated 4–7 days later with the B7H3 CAR-CXCR2 T cells or B7H3 T cells (control) and followed weekly with bioluminescent imaging.ResultsIrradiated (10 Gy) sarcoma cells express 2-9x higher IL-8 than non-irradiated sarcoma. T cells were transduced with efficiencies of 60–90%. INF-γ production was equivalent between the B7H3 CAR-T2a-CXCR2 T cells and B7H3 CAR T cells, but IL-2 production was significantly higher in the dual expressing CAR T cells. In vitro cytotoxicity with sarcoma spheroids was measured by Incucyte and showed faster and greater killing by B7H3 CAR-T2a-CXCR2 T cells than B7H3 CAR T cells. Furthermore, when sarcoma tumor bearing mice were treated with B7H3 CAR-T2a-CXCR2 T cells, tumors resolved completely by 4–5 weeks and had long-lasting remission.ConclusionsChemokine receptor expressing CAR T cells showed superior cytokine production and T cell activation/cytotoxicity compared to a CAR T construct alone. These finding lead to better efficacy in animal models and suggest a promising approach for pediatric sarcoma.ReferencesLuetke A, Meyers PA, Lewis I, Juergens H. Osteosarcoma treatment - where do we stand? A state of the art review. Cancer Treat Rev 2014;40:523–32.Bleyer A, Barr R, Hayes-Lattin B, et al. The distinctive biology of cancer in adolescents and young adults. Nat Rev Cancer 2008;8:288–98.Buechner J, SA G, SL M, et al. Global Registration Trial of Efficacy and Safety of CTL019 in Pediatric and Young Adult Patients with Relapsed/Refractory (R/R) Acute Lymphoblastic Leukemia (ALL): Update to the interim analysis. 2017 European Hematology Association Annual Meeting: Madrid, Spain2017.Maude SL, Laetsch TW, Buechner J, et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med 2018;378:439–48.Gill S, Maus MV, Porter DL. Chimeric antigen receptor T cell therapy: 25 years in the making. Blood Rev 2016;30:157–67.Fousek K, Ahmed N. The Evolution of T-cell Therapies for Solid Malignancies. Clin Cancer Res 2015;21:3384–92.Newick K, Moon E, Albelda SM. Chimeric antigen receptor T-cell therapy for solid tumors. Mol Ther Oncolytics 2016;3:16006.Nagarsheth N, Wicha MS, Zou W. Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy. Nat Rev Immunol 2017;17:559–72.Ethics ApprovalThe animal experiments discussed in the abstract were approved by the University of Colorado IACUC, protocol #00251.
IKZF3 deficiency potentiates chimeric antigen receptor T cells targeting solid tumors