Comparison of seven CD19 CAR designs in engineering NK cells for enhancing anti‐tumour activity

Abstract

AbstractChimeric antigen receptor‐natural killer (CAR‐NK) cell therapy is emerging as a promising cancer treatment, with notable safety and source diversity benefits over CAR‐T cells. This study focused on optimizing CAR constructs for NK cells to maximize their therapeutic potential. We designed seven CD19 CAR constructs and expressed them in NK cells using a retroviral system, assessing their tumour‐killing efficacy and persistence. Results showed all constructs enhanced tumour‐killing and prolonged survival in tumour‐bearing mice. In particular, CAR1 (CD8 TMD‐CD3ζ SD)‐NK cells showed superior efficacy in treating tumour‐bearing animals and exhibited enhanced persistence when combined with OX40 co‐stimulatory domain. Of note, CAR1‐NK cells were most effective at lower effector‐to‐target ratios, while CAR4 (CD8 TMD‐OX40 CD‐ FcεRIγ SD) compromised NK cell expansion ability. Superior survival rates were noted in mice treated with CAR1‐, CAR2 (CD8 TMD‐ FcεRIγ SD)‐, CAR3 (CD8 TMD‐OX40 CD‐ CD3ζ SD)‐ and CAR4‐NK cells over those treated with CAR5 (CD28 TMD‐ FcεRIγ SD)‐, CAR6 (CD8 TMD‐4‐1BB CD‐CD3ζ 1‐ITAM SD)‐ and CAR7 (CD8 TMD‐OX40 CD‐CD3ζ 1‐ITAM SD)‐NK cells, with CAR5‐NK cells showing the weakest anti‐tumour activity. Increased expression of exhaustion markers, especially in CAR7‐NK cells, suggests that combining CAR‐NK cells with immune checkpoint inhibitors might improve anti‐tumour outcomes. These findings provide crucial insights for developing CAR‐NK cell products for clinical applications.