Effectiveness of CAR-T cells engineered with TGF-βr/IL-9R domain-swapped chimeric receptor and membrane-bound IL-2 for treatment of CEA+ solid tumors.

Abstract

e14502 Background: Despite recent progress, CAR-T therapy for solid tumors remains limited by poor infiltration, an immunosuppressive tumor microenvironment (TME), and inadequate pro-survival signaling. Existing strategies, including TGF-β blockade or IL-2 supplementation, cause substantial non-target toxicity. Here, we engineered domain-swapped chimeric receptor (DSCR) that convert TGF-β’s immunosuppressive signals into T cell proliferative/survival signals. Screening identified TGF-βR/IL-9R DSCR, composed of TGF-βR extracellular and IL-9Rγ intracellular domains, which triggers STAT5 phosphorylation and activate the IL-9 pathway. Integration of TGF-βR/IL-9R DSCR with mbIL-2 further generated Super-empowered CAR-T cells (SE-CAR), which preserve effector phenotype, promote boosts expansion, and enhance antitumor efficacy in solid tumors. Methods: We first assessed the activation status and expansion capability of SE-CAR T cells. Serial killing assays were subsequently performed to evaluate SE-CAR T cell durability by measuring their capacity for repeated tumor target elimination. On this basis, we evaluated SE-CAR-T cells’ tolerance and responsiveness to TGF-β1 in a mimicked TME. Moreover, the supernatant of each round was used to detect the release of cytokines. Finally, the in vivo expansion and antitumor efficacy of SE-CAR T cells were validated in cecal and lung orthotopic tumor models, rectal and gastric cancer subcutaneous models. Results: SE-CAR cells significantly increased the proportion of phosphorylated STAT5 in response to TGF-β1 (42.00 ± 10.71 vs 8.33 ± 3.40) %. Moreover, SE-CAR T cells exhibited superior serial killing and proliferative capacity compared with control CAR-T cells (8 vs 5 rounds). Similarly, SE-CAR T cells showed higher cytokine secretion at 5th sequential stimulation (30448 pg/mL vs 11505 pg/mL). Furthermore, SE-CAR T cells retained proliferative capacity and exhibited enhanced cytotoxicity activity in response to TGF-β1. Then, we further evaluated the antitumor efficacy of SE-CAR in four xenograft mice models. At 14 days post CAR-T cell infusion, the SE-CAR T cells exhibited significantly higher CAR copy numbers than control CAR-T cells (Table1). In addition, the tumor fluorescence curves also showed that SE-CAR T cells possess more prominent antitumor efficacy in vivo . Conclusions: These preclinical studies highlight the promise of SE-CAR as a next-generation multifunctional CAR-T modality, with the capacity to preserve effector phenotype, boost T-cell fitness and expansion, and improve TME tolerance for the treatment of CEA+ solid tumors. Mouse model CAR T SE-CAR T / Days Mean ± S.D. cecal orthotopic tumor model D14 944±1681 52516±81593 lung orthotopic tumor model D14 25951±10825 233744±65929 rectal cancer model D14 5697±9237 185814±170118 gastric cancer model D14 899±960 30214±30259