A novel rapid Th9/Tc9-polarized CAR-T manufacturing platform to enable metabolic fitness and superior antitumor potency in solid tumors.

Abstract

e14501 Background: The efficacy of CAR-T therapy in solid tumors is hindered by poor T-cell fitness and rapid exhaustion within the tumor microenvironment (TME). IL-9-secreting Th9/Tc9 subsets possess superior adaptability and persistence compared to canonical Th1/Tc1 cells. We present ToughCAR, a rapid manufacturing platform generating Th9/Tc9-polarized CAR-T cells with enhanced metabolic fitness and antitumor potency. Methods: A head-to-head comparison was performed between CEACAM5-specific C-CAR (conventional) and ToughCAR generated from healthy donors and patients. Assessments included flow cytometric, cytotoxicity, and cytokine assays. Metabolic fitness and exhaustion resistance were evaluated via repeat antigen stimulation under hypoxia, with transcriptional profiling (bulk RNA-seq) at pre-stimulation, Round 1, and Round 3. In vivo efficacy and pharmacokinetics were investigated in NCG mice bearing N87 xenografts treated with C-CAR or ToughCAR at escalating doses (0.5–2×10⁶ cells). Results: The 7-day ToughCAR process achieved Th9/Tc9 polarization. Phenotypically, ToughCAR enriched stem-like memory subsets (Tscm: 91.6% vs 77.1%, p = 0.0206) and minimized differentiation (CD197-: 0.6% vs 18.9%, p = 0.005). Functionally, ToughCAR exhibited 4–7-fold higher secretion of IL-2 and TNF-α, while maintaining comparable IFN-γ levels and cytotoxicity relative to C-CAR. Under repeat stimulation, ToughCAR exhibited > 20-fold greater proliferation. RNA-seq revealed metabolic rewiring in ToughCAR cells, characterized by upregulated oxidative phosphorylation and glycolysis pathways, enabling sustained fitness. In vivo, ToughCAR displayed superior dose-dependent expansion (Table). At 1×10⁶ cells, ToughCAR achieved ≈10-fold higher Cmax (p = 0.0021) and ≈8-fold higher AUC 0-56 (p = 0.0012) compared to C-CAR. Notably, superior expansion and persistence were maintained even at the lowest dose (0.5×10⁶). This robust expansion translated into complete tumor regression at doses1×10⁶, with cured mice rejecting secondary tumor rechallenge. Conclusions: ToughCAR establishes a novel manufacturing paradigm by generating metabolically plastic, exhaustion-resistant Th9/Tc9 cells. By overcoming exhaustion and ensuring robust expansion, ToughCAR addresses critical barriers in solid tumor therapy. Crucially, this superior potency translates into clinical benefit, supported by a promising 57% objective response rate (ORR) in patients with heavily pre-treated colorectal cancer in our ongoing first-in-human study. Pharmacokinetics of CAR-T cells in xenograft model (n=5 per group). Dose Level (Cells) Group Mean AUC 0-56d (copies × days) Mean Cmax (copies/μg) 0.5 × 10⁶ C-CAR 3.08 × 10⁴ 1.86 × 10³ ToughCAR 3.85 × 10⁵ 1.45 × 10⁴ 1 × 10⁶ C-CAR 5.73 × 10⁵ 2.99 × 10⁴ ToughCAR 4.38 × 10⁶ 2.85 × 10⁵ 2 × 10⁶ C-CAR 9.47 × 10⁵ 5.71 × 10⁴ ToughCAR 7.34 × 10⁶ 3.70 × 10⁵