Frontiers in immunology · 2026
Clinical-scale 10-day TCR-T cell manufacturing using IL-2/7/15 and TGF-β promotes early memory and tissue-resident-like phenotypes and robust antitumor activity in vitro.
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Shih Yi-Ping, Drokin Stephan, Burke Olivia, Huang Huayu, Leung Amy, Bravo-Manriquez Marco, Jang Myungkyu, Syrkina Marina S, Julian Laura, Sanjuan Nelson, Tran Eric
Аннотация
BACKGROUND: Adoptive cell therapy (ACT) using TCR-engineered T (TCR-T) cells is a promising strategy for treating solid tumors. One factor that influences the efficacy of ACT is the type of T cells used, with T cells displaying younger, less differentiated or tissue resident phenotypes associated with greater antitumor activity. We aimed to develop a rapid, clinical-scale protocol to generate younger and more potent TCR-T cells for therapy.
METHODS: Patient-derived PBMC were stimulated, CD8+ enriched, retrovirally transduced to express KRAS G12D-targeting TCRs, and expanded for 10 days in the presence of a novel cytokine cocktail (CKT) containing IL-2, IL-7, IL-15, and TGF-β. The impact of CKT on the phenotype, effector function, and in vitro antitumor activity was evaluated and compared to TCR-T cells manufactured with IL-2. This process was then adapted for clinical-scale manufacturing.
RESULTS: TCR-T cells generated with CKT displayed an increased frequency of early memory (Tn/scm) and tissue-resident (Trm)-like T cells with decreased KLRG1 expression compared to IL-2 manufactured TCR-T cells. CKT manufactured TCR-T cells demonstrated higher 4-1BB upregulation, IFN-γ, TNF, and granzyme B (GZMB) production, and enhanced killing of pancreatic and colorectal cancer cell lines in 2D and 3D tumor spheroid co-culture. Clinical-scale engineering runs yielded 3.30 and 6.15 x 109 total cells that displayed similar phenotypic and functional attributes observed in the small-scale studies.
CONCLUSION: Our novel 10-day TCR-T manufacturing protocol using IL-2, IL-7, IL-15, and TGF-β generates TCR-T cells characterized by distinct memory and tissue residency markers such as CCR7, CD103, and CD49a, and potent effector functions with the potential to improve the efficacy of adoptive cell therapy.
METHODS: Patient-derived PBMC were stimulated, CD8+ enriched, retrovirally transduced to express KRAS G12D-targeting TCRs, and expanded for 10 days in the presence of a novel cytokine cocktail (CKT) containing IL-2, IL-7, IL-15, and TGF-β. The impact of CKT on the phenotype, effector function, and in vitro antitumor activity was evaluated and compared to TCR-T cells manufactured with IL-2. This process was then adapted for clinical-scale manufacturing.
RESULTS: TCR-T cells generated with CKT displayed an increased frequency of early memory (Tn/scm) and tissue-resident (Trm)-like T cells with decreased KLRG1 expression compared to IL-2 manufactured TCR-T cells. CKT manufactured TCR-T cells demonstrated higher 4-1BB upregulation, IFN-γ, TNF, and granzyme B (GZMB) production, and enhanced killing of pancreatic and colorectal cancer cell lines in 2D and 3D tumor spheroid co-culture. Clinical-scale engineering runs yielded 3.30 and 6.15 x 109 total cells that displayed similar phenotypic and functional attributes observed in the small-scale studies.
CONCLUSION: Our novel 10-day TCR-T manufacturing protocol using IL-2, IL-7, IL-15, and TGF-β generates TCR-T cells characterized by distinct memory and tissue residency markers such as CCR7, CD103, and CD49a, and potent effector functions with the potential to improve the efficacy of adoptive cell therapy.