Chimeric 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.

Background: Patients with acute myeloid leukemia (AML) are often older, which brings challenges of endurance and persistent efficacy of autologous chimeric antigen receptor (CAR)-T cell therapies. Allogenic CAR-natural killer (NK) cell therapies may offer reduced toxicities and enhanced anti-leukemic potential against AML. CD33 CAR-NK cells have been investigated for AML therapy. However, the fratricide-mediated lysis of CD33-expressing NK cells by CD33 CAR-NK cells limits the expansion and efficacy of CD33 CAR-NK cells. Mesothelin (MSLN), a tumor differentiation antigen, is highly expressed in a fraction of patients with AML, making it a promising target for AML therapy. Methods: We designed a novel CD33-MSLN Loop CAR (Loop CAR) and evaluated its antitumor efficacy in human umbilical cord blood-derived NK (UCB-NK) cells and human pluripotent stem cell-derived NK (hPSC-iNK) cells. To further avoid fratricide caused by endogenous CD33 expression in NK cells, we established an hPSC-derived cell line via knockout of the CD33 gene (CD33^KO) and engineered Loop CAR. We generated CD33^KO-Loop CAR-iNK cells using an organoid induction approach. The efficacy of CD33^KO-Loop CAR-iNK cells against tumor cells expressing CD33 and MSLN was investigated both in vitro and in AML xenograft mice. Results: Loop CAR-NK cells exhibited superior cytotoxicity against dual-antigen-positive tumor cell lines and primary AML cells compared with CD33 CAR-NK and MSLN CAR-NK cells. Moreover, Loop CAR-NK cells showed upregulated signaling pathways related to NK cell activation and cytotoxic function. The loss of CD33 in iNK cells effectively avoided fratricide, improved expansion ability, and significantly enhanced CD33 and MSLN-mediated specific cytotoxicity of Loop CAR-iNK cells. Moreover, the CD33^KO-Loop CAR-iNK cells demonstrated superior tumor-killing activity in AML xenograft mouse models and significantly prolonged mouse survival. Conclusion: Loop CAR empowered both UCB-NK cells and hPSC-iNK cells with superior cytotoxicity against CD33⁺MSLN⁺ tumor cells. Genetic disruption of CD33 avoided fratricide and improved efficacy of Loop CAR-iNK cells against AML. This innovative strategy possesses unique advantages and translational potential for treating AML.

The lack of persistence and short-term efficacy presents a major challenge for CAR-NK cell therapy. Here, we addressed this issue by developing pluripotent stem cell-derived NK lineage-committed progenitor (iNKP) cell therapy. For the first time, we generated abundant iNKP cells via an organoid culture system. The iNKP cells, engineered to express CXCR4 and chimeric antigen receptors (CAR), efficiently migrated to the bone marrow and generated CAR-iNK cells persisting in peripheral blood (PB) for over 80 days. Notably, CAR-iNKP cell therapy durably protected animals from tumour occurrence. Furthermore, a single low-dose infusion of CAR-iNKP cells following conventional chemotherapy eradicated minimal residual disease (MRD), leading to long-term complete remission. Our findings present a novel strategy to overcome the limitations of traditional CAR-NK cell therapy and offer dural breakthroughs for the prevention of tumour occurrence and relapse.

Hoxb5 exhibits preferential expression in hematopoietic stem cells (HSCs) and uniquely marks the long-term HSCs (LT-HSCs). Previous studies have demonstrated the remarkable capability of Hoxb5 to alter cell fates when enforced expression in blood progenitors, such as B cell progenitors and multipotent progenitors. Additionally, Hoxb5 deficiency does not hinder the generation of LT-HSCs. However, the specific impact of Hoxb5 deletion on LT-HSCs has remained unexplored. To address this, we developed a conditional Hoxb5 knockout-reporter mouse model, wherein Hoxb5 was knock out by the Vav-cre recombinase, and the endogenous Hoxb5 promoter drove the expression of the blue fluorescent protein (BFP). Our findings revealed that the primary recipients, who transplanted with HSCs indicating Hoxb5 deficiency by the presence of BFP (BFP-positive HSCs), exhibited comparable levels of donor chimerism and lineage chimerism to recipients transplanted with HSCs that spontaneously did not express Hoxb5 and thus lacked BFP expression (BFP-negative HSCs). However, during the secondary transplantation, recipients receiving total bone marrow (BM) from the primary recipients with BFP-positive HSCs showed significantly higher levels of donor chimerism and more robust multi-lineage chimerism compared to those receiving total BM from the primary recipients with BFP-negative HSCs. Our results indicate that deleting Hoxb5 in LT-HSCs transiently influences their lineage differentiation bias without compromising their long-term self-renewal capacity. These findings highlight the primary role of Hoxb5 in regulating lineage commitment decisions in LT-HSCs, while emphasizing that its presence is not indispensable for the maintenance of long-term self-renewal capacity.