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Aim: The aim of the current study was to fully elucidate the functions of T cells genetically modified with an erbB2-specific chimeric antigen receptor (CAR)., Material & Methods: In this study, key functional parameters of CAR T cells were examined following antigen-specific stimulation of the chimeric anti-erbB2 receptor., Results: Gene-modified T cells produced the cytokines IFN-γ, IL-2, IL-4, IL-10, TNF-α and IL-17, and the chemokine RANTES upon CAR ligation. A multifunctional capacity of these CAR T cells was also demonstrated, where 13.7% of cells were found to simultaneously express IFN-γ and CD107a, indicative of cytolytic granule release. In addition, the CAR T cells were able to respond to a greater degree on the second ligation of CAR, which has not been previously shown. IFN-γ secretion levels were significantly higher on second ligation than those secreted following initial ligation. CAR-expressing T cells were also demonstrated to lyze erbB2-expressing tumor cells in the absence of activity against bystander cells not expressing the erbB2 antigen, thereby demonstrating exquisite specificity., Conclusion: This study demonstrates the specificity of CAR gene-engineered T cells and their capacity to deliver a wide range of functions against tumor cells with an enhanced response capability after initial receptor engagement.

Adoptive immunotherapy is a promising approach for the treatment of cancer; however, autoimmunity against normal tissue can be a serious complication of this therapy. We hypothesized that T-cell cultures responding maximally only when engaging two antigens would be more specific for tumor cells, and less active against normal cells, as long as the tumor expressed both antigens, while normal cells expressed only one of the antigens. A model system was developed consisting of cell lines expressing either folate binding protein or erbB-2, representing 'normal' tissue, and cells expressing both antigens representing tumor tissue. Human T-cell cultures were produced using two chimeric antigen receptor vectors ('dual transduced'), or using a single chimeric antigen receptor vector (monospecific). Dual-transduced T cells responded less against 'normal' cells compared with tumor cells. This relatively simple procedure produced T-cell cultures that were as active against a tumor as the monospecific cultures used traditionally, but had lower activity against model normal cells.