Human CXCR1 mRNA

A fundamental reason why current chimeric antigen receptor (CAR) immunotherapies fail to adequately treat solid tumors is the lack of infiltration of CAR immune cells into tumors. To address this issue, researchers electroporated natural killer (NK) cells with two mRNA constructs encoding the chemokine receptor CXCR1 and a CAR targeting a tumor-associated NKG2D ligand. CXCR1-modified NK cells exhibited increased migration into tumor supernatants in vitro and enhanced infiltration of human tumors in vivo in subcutaneous and intraperitoneal xenograft models. Most importantly, the cytotoxicity of CAR-NK cells was not affected by CXCR1 transgene expression, and enhanced tumor trafficking after intravenous injection resulted in significantly enhanced antitumor responses in mice bearing established peritoneal ovarian cancer xenografts. Together, these findings suggest that co-expression of CXCR1 and CAR may provide a novel strategy to enhance the efficacy of NK cells in solid cancer treatment.

Here, researchers transfected NK cells with mRNA encoding CXCR1 by electroporation to restore its expression. mRNA electroporation induced CXCR1 overexpression in more than 95% of NK cells (Figure 1D). Median fluorescence intensity (MFI) increased from undetectable levels on mock-electroporated NK cells to 15,000 on CXCR1-transfected cells. CXCR1 expression levels were approximately 3-fold higher in CXCR1-electroporated NK cells compared to freshly isolated NK cells (Figure 1B). Transgene expression persisted for at least 72 hours, the longest time point detected (Figure 1E). The researchers then tested the in vitro migration of transfected NK cells into conditioned media collected from a panel of IL-8-secreting human cancer lines. As shown in Figure 1F, conditioned media was as effective or even stronger than the chemokine IL-8 in attracting CXCR1-modified NK cells, but not NK cells that were not CXCR1-modified (mock control). The migration capacity of CXCR1-modified NK cells was increased by approximately 5-fold compared to mock NK cells. Mock NK cells showed signs of migration toward conditioned media from head and neck cancer cell lines that secrete CXCL10, which may be due to the expression of CXCR3 after NK cell expansion. These results indicate that restoring CXCR1 expression on the ex vivo-expanded NK cells can effectively promote their migration toward IL-8-secreting cancer cells.

Figure 1. CXCR1 and CXCR2 Expression on Natural Killer (NK) Cells and Overexpression of CXCR1 to Restore NK Cell Migration Ability. (Ng Y Y, Tay J C K, Wang S., 2020)