Dual-targeting CD133/PD-L1 CAR-T plus αPD-1 overcomes immunosuppressive microenvironment and enhanced by radiation pre-conditioning.

Innovative chimeric antigen receptor (CAR) T cell designs and combinational approaches are needed for enhancing therapeutic effectiveness in solid tumors. We developed and assessed a novel dual-targeting CAR-T therapy that combines an αPDL1.CD28 chimeric receptor with a second-generation αCD133 CAR to target CD133(+) tumors. The αPDL1.CD28 structure activated the CD3ζ signaling in cis by clustering with αCD133 CAR via CD28 dimerization. Binding to programmed cell death ligand-1 (PD-L1) through αPD-L1 CAR improved the CD133-targeted cytotoxic function of T cells by enhancing activation signals and countering inhibitory signals. Combination with programmed cell death receptor-1 (PD-1) blockade further disrupted the PD-L1/PD-1 inhibitory signal, achieving prolonged therapeutic efficacy. Moreover, radiation pre-conditioning (10 Gy/1 fraction or 4 Gy/2 fractions) maximized the antitumor effects of CAR-T plus PD-1 blockade, inducing complete tumor regression in mice. Radiation induced a unique tissue-resident memory CAR-T cell phenotype with high CXCR6 and CD103 expression. As the ligand of CD103, E-cadherin expression increased in tumor cells after irradiation, potentially mediating E-cadherin-CD103 interactions between tumor cells and tissue-resident memory T cells. Our study introduces a novel dual-targeting CD133/PD-L1 CAR-T cell and further demonstrates the efficacy and rationale of the triple-combination approach in solid tumors.