Triple-costimulatory GD2-CAR T cells incorporating CD28, 4-1BB and CD27 mediate potent cytotoxicity and reduce exhaustion in solid tumour models.

Disialoganglioside (GD2) is a validated tumour-associated antigen for chimeric antigen receptor (CAR) T-cell therapy; yet most GD2-CAR constructs carry limited costimulatory signaling, which may constrain efficacy and durability. We engineered humanised hu3F8-based GD2-CAR T cells bearing dual or triple costimulatory domains- GD2-28BBζ (CD28/4-1BB), GD2-28.27ζ (CD28/CD27) and GD2-28BB27ζ (CD28/4-1BB/CD27) - and compared their in-vitro function against GD2-positive neuroblastoma (SK-N-AS), lung adenocarcinoma (A549) and retinoblastoma (Y79 and WERI-Rb-1) cells. All three mediated antigen-specific cytotoxicity against GD2-positive lines while sparing GD2-negative targets. At the lowest effector-to-target ratio, GD2-28BB27ζ T cells retained significantly killing of SK-N-AS cells where the third-generation constructs did not, indicating potent cytotoxicity under limiting effector conditions, whereas GD2-28.27ζ T cells showed the greatest proliferation, reaching significance against WERI-Rb-1. On antigen engagement, GD2-28BB27ζ T cells sustained cytotoxicity while secreting less IL-2, IFN-γ and TNF-α than the other constructs, indicating an uncoupling of lytic function from effector-cytokine output. Under serial tumour rechallenge, GD2-28BB27ζ T cells maintained cytolytic function, acquired fewer activation and exhaustion markers (CD69, TIM-3 and, versus GD2-28BBζ, LAG-3; PD-1 unchanged), better preserved their CAR⁺ population, and retained a larger naïve-like (CD45RO⁻CD62L⁺) subset than the other constructs. Together, these in-vitro findings indicate that combining CD28, 4-1BB and CD27 within a single CAR confers potent cytotoxicity with low effector-to-target ratios, lower effector-cytokine output, reduced exhaustion-marker acquisition and greater phenotypic durability, identifying GD2-28BB27ζ as a promising configuration for GD2-positive tumours that warrants in vivo evaluation.