Antigen-induced IL-12 potentiates piggyBac-engineered HER2-CAR-T cells against gastric cancer

Gastric cancer (GC) often progresses asymptomatically, resulting in late-stage diagnosis and limited therapeutic options, thus underscoring the urgent need for more effective treatments. Although chimeric antigen receptor (CAR) T-cell therapy demonstrates promise, its efficacy is frequently hindered by the immunosuppressive tumor microenvironment (TME). Interleukin-12 (IL-12), a potent enhancer of T-cell immunity capable of reprogramming the TME, was leveraged in this study by engineering a single-chain recombinant human IL-12 (rhIL-12) fusion protein (p40/p35) cloned into a piggyBac transposon plasmid under either the elongation factor-1α (EF-1α) promoter (PB-IL-12) for constitutive IL-12 secretion or the NFAT-IL-2 promoter (NFAT-IL-12) for antigen-inducible secretion. These IL-12 constructs were co-transfected with a HER2-specific CAR into human T cells, and puromycin-selected HER2-CAR-T cells were subsequently evaluated for antitumor efficacy in vitro and in vivo. In vitro, PB-IL-12-CAR-T cells exhibited significantly reduced proliferation compared to NFAT-IL-12-CAR-T cells or HER2-CAR-T cells lacking IL-12, with phenotypic analysis revealing constitutive IL-12 secretion drove enrichment of CD4⁺T cells and CD62L⁺central memory T cells. Functionally, HER2-CAR-T cytotoxicity and IFN-γ correlated with target-antigen density, whereas IL-12 secretion uniformly enhanced tumor-cell killing across both high- and low-antigen tumors. In vivo, IL-12 secretion augmented HER2-CAR-T antitumor activity, with PB-IL-12-CAR-T cells producing higher and more sustained systemic IL-12 and IFN-γ levels than NFAT-IL-12-CAR-T cells while achieving equivalent tumor control. The antigen-inducible system's regulated IL-12 secretion and preserved proliferative potential position it as a promising therapeutic strategy for refractory GC.