design and production an effective bispecific tandem chimeric antigen receptor on t cells against cd123 and folate receptor ß towards b-acute myeloid leukaemia blasts

Objective: the clinical studies of acute myeloid leukaemia (aml) revealed that antigen escaping variants cause cancer recurrence even after treatment with chimeric antigen receptor (car)-t cells that target a single tumour antigen. due to the heterogeneous expression of antigens on leukaemia blasts, we hypothesized that a novel bispecific car, directed to the folate receptor beta (frβ)-binding single-chain variable fragment (scfv) and an il3α-binding receptor (cd123) that has more expression in aml blasts, can decrease car-t cell exhaustion and increase the efficacy of car-t cells to prevent antigen escaping and consequent recurrence of aml. materials and methods: in this experimental study, the survival, proliferation, and cytolysis of car-t cells remains suboptimal even with a costimulatory endodomain. hence, we designed and constructed a tandem car that joins an frβ and cd123 in the second generation retroviral vector to generate a bispecific tandem car (tancar-t cell). results: tancar frβ-cd123 t cells showed distinct binding to frβ or cd123 expressing cells. they could lyse the leukaemia cell lines (66.1 ± 11%) comparable to the single car-t cells against these determinants. tancar frβ- cd123 t cells simultaneously engaged frβ and cd123, which promoted t cell activation in targeting and lysis of the examined leukaemia cell lines. tancar-t cell significantly induced interferon gamma (ifnγ) and interleukin 2 (il-2) production more than single car-t cells, which produced a synergistic enhancement of tancar frβ-cd123 t cell function when dual antigens faced simultaneously. conclusion: dual-specific tancar frβ-cd123 t cells showed therapeutic potential to improve aml control by coengaging frβ and cd123 molecules in a robust, divalent immune system. this strategy may be a useful therapeutic approach in patients with relapsed b-cell malignancies.