Trogocytosis is the intercellular transfer of membrane-associated molecules documented in different biological processes. This process has been extensively studied in immune system cells and can alter the immune response. Lymphocytes expressing a synthetic chimeric receptor (CAR-T cells) against tumor antigens have shown clinical results against several types B leukemias. Recently, it has been described that trogocytosis processes in CAR-T cells, which acquire the tumor antigen from the target cell, may have a negative impact on this antitumor therapy.

Our aim is to characterize trogocytosis process in an in vitro model by using T cells transduced with a CAR against the pan-ErbB molecules.

Expression of CAR with 41BB as co-stimulatory domain and directed against ErbB molecules, is obtained by lentiviral transduction. Target cells are cell lines derived from HNSCC patients (VU-1131 and VU1365) transfected to express GFP/Luc and overexpressing ErbB molecules. CAR-T cells are added to the adherent lines at 5:1 and 1.25:1 (E:T) ratios and the percentage of lymphocytes acquiring ErbB molecules that are Trog⁺ (CD3⁺ErbB⁺EGFP⁻), Trog⁻ (CD3⁺ErbB⁻EGFP⁻) and tumor cells (CD3⁻ErbB⁺EGFP+) has been analysed at different time points in CAR⁺ and CAR⁻ subpopulations. The intensity of trogocytosis is analysed by MFI of ErbB.

Trogocytosis is evident in CAR-expressing lymphocytes after 24 hours of interaction with target cells. Notably, troghigh lymphocytes show significantly higher levels of fratricide killing compared to troglow lymphocytes associated to reduction in the level of ErbB1 in target cells. Trogocytosis could impair the anti-tumor gene therapy of this CAR immunotherapy.