Background: Patients with advanced sarcoma have limited treatment options and poor survival. In our phase 1 dose-escalation trial, intravenous administration of up to 1x108/m2autologous HER2-CAR T cells in patients with HER2+ sarcoma was safe. The goal of the current study is to evaluate if lymphodepleting chemotherapy can safely improve the expansion of HER2-CAR T cells. Methods: In a phase 1 clinical trial, NCT00902044, we administered 1x108/m2autologous HER2-CAR T cells to patients with recurrent/refractory HER2+ sarcoma following lymphodepletion. Results: Ten patients with refractory/metastatic HER2+ sarcoma (5 osteosarcoma, 3 rhabdomyosarcoma [RMS], 1 Ewing sarcoma, 1 synovial sarcoma) with a median age of 14 (range: 4 to 54) were treated; one RMS patient was re-enrolled and retreated. Each patient received up to 3 infusions of 1x108cells/m2 CAR T cells after lymphodepletion with either fludarabine (Flu; n = 3) or Flu and cyclophosphamide (Flu/Cy; n = 8). Eligible patients were given up to an additional 5 subsequent infusions without lymphodepletion. Flu and Flu/Cy induced lymphopenia with an absolute lymphocyte count of Conclusions: Administration of lymphodepletion chemotherapy followed by autologous HER2-CAR T cells is safely tolerated and is associated with objective clinical benefit in some patients with advanced HER2+ sarcoma. Immune correlative studies suggest that the HER2-CAR T cells given in combination with Flu/Cy lymphodepletion induce endogenous immune reactivity. These findings warrant further evaluation in a phase 2 study as a single agent or in combination with other approaches. Citation Format: Shoba A. Navai, Christopher Derenzo, Sujith Joseph, Khaled Sanber, Tiara Byrd, Huimin Zhang, Melinda Mata, Claudia Gerken, Ankita Shree, Pretty R. Mathew, Olga Dakhova, Vita Salsman, John Hicks, Zhongzhen Yi, Meng-Fen Wu, Tao Wang, Bambi Grilley, Cliona Rooney, Malcom Brenner, Helen Heslop, Adrian Gee, Stephen Gottschalk, Nabil Ahmed, Meenakshi Hegde. Administration of HER2-CAR T cells after lymphodepletion safely improves T cell expansion and induces clinical responses in patients with advanced sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-147.