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TGFβ-resistant TGFBR2 KO CAR-NK cells manufactured by a one-step scalable process show enhanced efficacy against solid tumors
Abstract No. : 76

Category

Engineered innate immune cell therapy (e.g., NK,NKT, macrophage,γδT, etc.)
TGFβ-resistant CAR-engineered natural killer (NK) cells represent a promising off-the-shelf strategy for solid tumor immunotherapy, but their efficacy is often limited by the immunosuppressive tumor microenvironment, particularly TGFβ. Here, we developed a virus-free, clinically scalable one-step electroporation platform to generate mesothelin (MSLN)-targeting, TGFβ-resistant CAR-NK cells by simultaneously delivering Cas9 ribonucleoproteins and a linear dsDNA HDR donor to knock out TGFBR2 and knock in the CAR at the TGFBR2 locus. Compared with a conventional two-step AAV donor delivery workflow, optimization of sgRNA/donor design and electroporation conditions improved knock-in efficiency and CAR expression. Importantly, transient dexamethasone (Dex) exposure during manufacturing increased nuclear donor DNA availability and enhanced allele-level CAR integration. These changes translated into stronger cytotoxicity against AsPC-1 pancreatic cancer cells and patient-derived pancreatic cancer organoids. Multi-omics analyses further showed that Dex conditioning was associated with increased oxidative phosphorylation and ATP-generation programs, together with targeted chromatin accessibility changes consistent with improved metabolic fitness of NK cells. In a xenograft pancreatic ductal adenocarcinoma (PDAC) mouse model, Dex-treated one-step TGFBR2 KO/KI CAR-NK cells achieved superior suppression of solid tumor growth. Together, these results support a practical manufacturing strategy for metabolically fortified, TGFβ-resistant CAR-NK cells and highlight their therapeutic potential for solid tumors.
#CAR-NK
#Knock-in
#Dexamethasone
#TGFBR2
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