Abstract Background: Patients with anaplastic or poorly differentiated recurrent thyroid carcinomas have a poor prognosis, with a median survival of < 1 year. Previously, we have shown a significant correlation between ICAM-1 overexpression and malignancy in thyroid cancer and have pioneered the use of ICAM-1 targeted CAR T cells as a novel treatment modality. For clinical translation, we designed CAR T cells possessing micromolar affinity to ICAM-1 to avoid cytotoxicity in normal cells with basal levels of ICAM-1 expression. Here, we report the automated process of CAR T cell manufacturing with CliniMACS Prodigy (Miltenyi Biotec) using cryopreserved peripheral blood leukopak cells as starting material. Methods: We have chosen to use CliniMACS Prodigy system for a closed, automated, and robust CAR T cell manufacturing process. Using a cryopreserved leukopak, TCT v2.0 protocol began with rapidly thawing the leukopak while directly welded to the Prodigy. T cell lentivirus transduction was performed 2x, 24 and 48 hr post activation. CAR T cells were released after ~10 days of culture and cryopreserved in ready-to-infuse formulation (AIC100). Products were then subject to in vitro cytotoxicity, in vivo efficacy and safety in xenografted mice with ATC cells (8505C), and PET/CT imaging using 18F-NOTA-octreotide to detect T cell dynamics. Results: Prodigy manufactured CAR T cells were subjected to qualification and functional assays (n=7): cell viability, cell number, CAR expression, virus copy number (VCN), T cell subset and phenotype, and E to T assays. Consistent with reported values for Prodigy, our manufacturing protocol produced 55±9% transduction rate, 96±1.8% viability, and 2.9±0.7x10^9 final cell number. The VCN was in the range of 1.0-2.1, below the criteria of < 5 copies per cell. In NSG mice xenografted with 8505C ATC cells and treated with CAR T cells (1X= 1 million live CAR T maximum tolerable dose (MTD)= 10 million live CAR T) or non-transduced (NT) T cells, tumor burden was quantitatively evaluated by whole body luminescence imaging. Compared to cohorts with no treatment (NoT) or NT, the cohorts of AIC100 exhibited complete or near-complete tumor elimination lasting variable times before tumor relapse was seen in some subjects. Median survival time for NoT and NT groups were similar (38.5 vs. 42 days), while it was significantly longer for AIC100 1X and MTD groups (72 vs. >100 days). Conclusions: Selective anti-tumor activity in the absence of toxicity provides proof-of-concept that micromolar affinity tuned CAR T cells can be used to target tumors expressing high levels of antigen while avoiding normal tissues expressing basal levels of the same antigen. These studies support the initiation of a phase I study to examine the safety and potential efficacy of micromolar affinity tuned CAR T cells against newly diagnosed anaplastic and refractory or recurrent thyroid cancers. Citation Format: Yogindra Vedvyas, Jaclyn E. McCloskey, Yanping Yang, Irene M. Min, Thomas J. Fahey, Yen-Michael S. Hsu, Jing-Mei Hsu, Koen V. Besien, Ian Gaudet, Ping Law, Joon Kim, Eric V. Hofe, Moonsoo M. Jin. Clinical manufacturing of CAR T cells targeting ICAM-1 for a phase I study against advanced thyroid cancer therapy [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 2329.