Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

Abstract Background Head and neck squamous cell carcinoma (HNSCC) remains difficult to treat due to heterogeneous antigen expression and an immunosuppressive tumor microenvironment. The epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific splice form with no detectable protein expression in normal tissues. Nevertheless, its low and often heterogeneous expression in HNSCC has limited its utility as target antigen for immunological therapies using chimeric antigen receptor (CAR) T-cells. In contrast, full-length EGFR is frequently overexpressed in HNSCC, but its expression in normal tissues prevents its use as a directly activating CAR target. Thus, we investigated here whether EGFR can instead provide additional target antigen engagement as a non-signaling CAR (nsCAR) for EGFRvIII CARs. The nsCAR was derived from Cetuximab, an EGFR-binding monoclonal antibody whose epitope is retained in EGFRvIII and was co-expressed with different EGFRvIII-specific signaling CAR to investigate whether additional receptor engagement could enhance target-cell recognition and cytotoxicity. Methods Primary human T-cells were engineered to express EGFRvIII-specific CARs alone or in combination with EGFR-directed nsCARs. Functional activity was evaluated using in vitro cytotoxicity assays, time-resolved killing dynamics, and fluorescence microscopy to assess immunological synapse (IS) formation in HNSCC cell lines with defined levels of EGFRvIII expression. Results EGFRvIII CAR T-cells exhibited clear target antigen density–dependent cytotoxicity. Co-expression of the EGFR nsCAR specifically enhanced tumor cell killing in selected CAR and target antigen combinations, with the strongest effects observed under conditions of limited baseline CAR activity, whereas the nsCAR alone was non-cytotoxic. Fluorescence imaging revealed co-localization of CAR and nsCAR molecules at the T-cell–target-cell contact interface. Conclusions EGFR nsCAR co-expression can provide a modular strategy to enhance EGFRvIII CAR T-cell cytotoxicity under conditions of limited target antigen availability. These findings provide an in vitro proof of concept for additional target engagement through a non-signaling receptor and warrant further evaluation in more complex preclinical models.