Anti-PD-L1 scFv-secreting fifth-generation GD2-CAR T cells enhance antitumor function against GD2-low retinoblastoma.

Retinoblastoma (RB) is an aggressive pediatric intraocular malignancy in which chemoresistant or relapsed disease often requires enucleation, leading to vision loss. GD2-targeted chimeric antigen receptor (CAR) T cells are promising for GD2-positive tumors, but PD-L1-mediated immunosuppression and reduced surface antigen density can limit their activity. We developed a fifth-generation anti-GD2 CAR (GD2-CAR5), defined here as a fourth-generation backbone that additionally drives secretion of an anti-PD-L1 single-chain variable fragment (scFv) from the same transgene cassette, incorporating a humanized 3F8 anti-GD2 scFv and CD28, 4-1BB, CD27, and CD3ζ signaling domains. CAR surface expression and scFv secretion were confirmed in primary transduced T cells, and the secreted scFv bound and blocked PD-L1 on target cells during co-culture. Against both high- and low-antigen-density RB cells, GD2-CAR5 and fourth-generation GD2-CAR4 T cells mediated comparable cytotoxicity and secreted comparable levels of granzyme B and IFN-γ; however, only GD2-CAR5 T cells were cytotoxic at the lowest effector-to-target ratio tested against low-density targets. Under repeated antigen re-stimulation, GD2-CAR5 T cell numbers remained above baseline through day 12, whereas GD2-CAR4 T cell numbers declined, without increased exhaustion marker expression. These findings provide