Hitoshi Kiyoi

Chimeric antigen receptor T (CAR-T) cells targeting multiple antigens (Ag), may reduce the risk of immune escape following the loss of the target Ag and further increase the efficacy of treatment. We developed dual-targeting CAR-T cells that target CD19 and CD37 Ags and evaluated their antitumor effects. CD19/CD37 dual CAR-T cells were generated using cotransduction and simultaneous gene transfer of two types of lentiviral vectors transferring CD19CAR or CD37CAR genes, including the intracellular domains of CD28 and CD3ζ signaling domains. These dual CAR-T cells contained three fractions: CD19/CD37 bispecific CAR-T cells, single CD19CAR-T cells, and single CD37CAR-T cells. In the functional evaluation of CAR-T cells in vitro, CD19/CD37 dual CAR-T cells showed adequate proliferation and cytokine production in response to CD19 and CD37 antigen stimulation alone or in combination. Evaluation of intracellular signaling revealed that dual CAR-T cell–mediated signals were comparable with single CAR-T cells in response to CD19- and CD37-positive B-cell tumors. Although the cytotoxicity of CD19/CD37 dual CAR-T cells in both CD19- and CD37-positive B-cell tumors was similar to that of single CD19 and CD37CAR-T cells, against CD19 and CD37 Ag-heterogeneous tumor, dual CAR-T cells demonstrated significantly superior tumor lysis compared with single CAR-T cells. Furthermore, CD19/CD37 dual CAR-T cells effectively suppressed Ag-heterogeneous Raji cells in a xenograft mouse model. Collectively, these results suggest that CD19/CD37 dual CAR-T cells may be effective target-Ag-loss B-cell tumor models in vitro and in vivo, which represents a promising treatment for patients with relapsed/refractory B-cell malignancies.