Clinical-scale 10-day TCR-T cell manufacturing using IL-2/7/15 and TGF-β promotes early memory and...

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.

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.

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.

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.