One-step knock-in CAR constructs in human NK cells enable scalable, TGFβ1-resistant immunotherapy for solid tumors

However, their efficacy in treating solid tumors is often compromised by the immunosuppressive tumor microenvironment (TME), where TGF-β1 plays a key role in inhibiting NK cell function. To address this challenge, our study focused on developing a simple, one-step process for generating CAR-NK cells, termed “one-step CAR-NK”, by simultaneously knocking out TGFβR2 and knocking in a CAR gene into human primary NK cells.

We first demonstrated that knocking out TGFβR2 using CRISPR-Cas9 enhanced NK-mediated cancer killing abilities with upregulated CD107a expression when co-cultured with AsPC-1 cells. Additionally, we optimized the donor DNA by inserting a binding motif for dexamethasone, which encodes a CAR targeting mesothelin (MSLN) as a tumor antigen, to integrate into the TGFβR2 locus. Using electroporation, the Cas9 protein/gRNA complex combined with CAR donor DNA significantly increased the knock-in efficacy, resulting in potent cancer killing against solid tumors.