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OBJECTIVES In accordance with the Precision Medicine Initiative, new treatment strategies for head and neck squamous cell carcinoma (HNSCC) are needed to yield better therapeutic outcomes. The purpose of this study was to establish and validate chimeric antigen receptor (CAR)-T cells targets in HNSCC. METHODS Putative CAR-T antigens were identified in The Cancer Genome Atlas database. To validate antigen suitability, quantitative RT-PCR, flow cytometry, and immunofluorescent staining were performed. A retroviral human CD70 CAR construct, using truncated CD27 conjugated with 4-1BB and CD3-zeta costimulatory molecules, was used to transduce activated human T cells to generate CD70 CAR-T cells. Cell-based cytotoxicity and cytokine ELISAs were used to measure efficacy of killing. RESULTS Nine potential CAR-T targets (CD276, EGFR, MICA, MICB, MAGE-A4, FAP, EPCAM, CD70, B4GALNT1) were identified based on their high expression in tumors compared to flanking control tissues. CD70 was selected for further proof-of-principle analysis based on its differential expression in several tumor subtypes, and showed substantial heterogeneity in individual tumors analyzed. Cell surface CD70 protein and CD70 mRNA were detected from low to high levels in established HNSCC cancer cell lines. CD70 was highly expressed in 4 of 21 tumor biopsies (19%), and 3 of 4 specimens showed strong CD70 expression on the tumor cell surface. CD70-specific CAR-T cells were generated and further demonstrated to recognize and kill CD70-positive HNSCC cells efficiently, but not CD70-negative cancer cells. CONCLUSION CD70-specific CAR-T cells specifically recognized and efficiently eliminated CD70-positive HNSCC cells. This study provides the basis for further investigation into CD70 and other CAR-T targets.