BACKGROUND: Cancer immunotherapy with transgenic T-cell receptor-engineered T cells (TCR-T) enables the targeting of intracellular tumor-specific antigens; in contrast, chimeric antigen receptor-modified T cells (CAR-T) mediate tumor cell killing via the recognition of surface antigens. In the case of acute myeloid leukemia, the lack of leukemia-specific surface antigens limits the efficacy of CAR-T cells; therefore, TCR-T cells may represent a more targeted immunotherapy approach. However, the tumor immunosuppressive environment eliminates the best-functioning, high-avidity TCR-T cells, thus creating a need for novel, enhanced TCR-T cells. METHODS: The piggyBac transposon vector used for gene modification of T cells expresses a T-cell receptor specific to the WT1 tumour antigen, an NFAT promoter-regulated CAR specific to GM-CSF receptor, a CD3xCD33 bispecific T-cell engager, and a truncated EGFR suicide gene system. The transgenic T cells were generated by electroporation using a single expression vector, and the efficiency of these engineered TCR-T cells was evaluated using models that utilized AML cell lines and primary AML cells. RESULTS: The NFAT-driven GM-CSF CAR significantly enhances the antileukemic activity of WT1-specific TCR-T cells, which importantly maintain specificity for their HLA/peptide antigenic complex. Next, by inserting the CD3xCD33 bispecific T-cell engager into the transposon vector, both TCR-T cells and recruited non-transfected bystander T cells can efficiently target the CD33 antigen, providing more robust antileukemic effects. CONCLUSION: The presented strategy, utilizing a single piggyBac transposon vector, enables the complex redirection of T-cell specificity against acute myeloid leukemia by inserting TCR, CAR, BiTE constructs, along with a tEGFR gene suicide system.

Faculty of Natural Sciences Charles University Prague Czechia

Institute of Hematology and Blood Transfusion Department of Immunomonitoring and flow cytometry Prague Czechia

Institute of Hematology and Blood Transfusion Department of Immunotherapy Prague Czechia

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