Simple SummaryDespite new therapeutic approaches in the last decades, prognosis and survival rates for head and neck squamous cell carcinomas (HNSCC) remain poor. Due to its high immune cell infiltration, immunotherapy has become a valuable tool for HNSCC, yet only three monoclonal antibodies have been approved to treat HNSCC. The aim of this study was to develop novel immunotherapeutic strategies for HNSCC employing CAR-NK cells that target HER1/epidermal growth factor receptor (EGFR), which is overexpressed in over 80% of HNSCC patients. We confirmed CAR-NK cell function by assessing cytotoxic killing, IFNγ secretion and CD107a degranulation in 2D and 3D co-culture models. Analyses of primary HNSCC cells that were still viable after anti-HER1 CAR-NK-92 cell challenge showed a high percentage of CD44v6-positive cells, indicating that targeting HER1 alone was not sufficient to eliminate this potential cancer stem cell population, which could contribute to disease progression such as metastasis. We conclude that CAR-NK cell therapy may be a useful tool for HNSCC treatment in combinatorial therapeutic regimens.Abstract(1) Background: HNSCC is a highly heterogeneous and relapse-prone form of cancer. We aimed to expand the immunological tool kit against HNSCC by conducting a functional screen to generate chimeric antigen receptor (CAR)-NK-92 cells that target HER1/epidermal growth factor receptor (EGFR). (2) Methods: Selected CAR-NK-92 cell candidates were tested for enhanced reduction of target cells, CD107a expression and IFNγ secretion in different co-culture models. For representative HNSCC models, patient-derived primary HNSCC (pHNSCC) cell lines were generated by employing an EpCAM-sorting approach to eliminate the high percentage of non-malignant cells found. (3) Results: 2D and 3D spheroid co-culture experiments showed that anti-HER1 CAR-NK-92 cells effectively eliminated SCC cell lines and primary HNSCC (pHNSCC) cells. Co-culture of tumor models with anti-HER1 CAR-NK-92 cells led to enhanced degranulation and IFNγ secretion of NK-92 cells and apoptosis of target cells. Furthermore, remaining pHNSCC cells showed upregulated expression of putative cancer stem cell marker CD44v6. (4) Conclusions: These results highlight the promising potential of CAR-NK cell therapy in HNSCC and the likely necessity to target multiple tumor-associated antigens to reduce currently high relapse rates.