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

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells offer a promising immunotherapy for various malignancies. 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. Furthermore, we explored a two-step CAR-NK manufacturing process, using AAV to deliver DNA instead of naked DNA. This method also showed high KI efficacy and robust cancer-killing capabilities. As with the one-step process, the supplement of dexamethasone further improved NK functions in two-step CAR-NK cells. Thus, our one-step CAR-NK platform, particularly in the presence of dexamethasone, holds promise as off-the-shelf therapeutics with a simple and scalable manufacturing process for treating solid tumors.