Introduction:
Chimeric antigen receptor (CAR) T-cell therapies are highly effective but carry risks like graft-versus-host disease (GvHD) and severe toxicities. Allogeneic natural killer (NK) cells offer a safer alternative, with lower toxicity and no GvHD risk. While CD19-directed CAR-NK cells show promise in hematological malignancies, their limited persistence and efficacy against solid tumors remain challenges. Conventional co-stimulatory domains (e.g., CD28, 4-1BB) enhance CAR-NK persistence, but achieving sustained tumor control requires optimized signaling. MyD88 and CD40, two unconventional signaling molecules, have shown promise in enhancing T-cell anti-tumor activity and may offer similar benefits for NK cells. MyD88 activates rapid, transient NF-κB signaling while CD40 drives slower, sustained NF-κB activation, promoting immune memory. Together, they balance NF-κB signaling to prevent NK cell exhaustion and enhance persistence and cytokine secretion. We hypothesize that incorporating MyD88/CD40 into CAR constructs will outperform conventional co-stimulation strategies in enhancing CAR-NK cell functionality.

Methods:
We engineered CAR constructs targeting TROP2 or CD70 with MyD88/CD40 signaling domains, individually and combined. These constructs were compared to CD28-based CARs for their ability to enhance NK cell proliferation, cytotoxicity, cytokine profiles, and long-term anti-tumor efficacy in vitro.

Results:
Preliminary data indicate that MyD88/CD40 or CD40 alone significantly enhances NK cytotoxicity, persistence, and cytokine secretion during repeated tumor challenges. These constructs show potential for durable anti-tumor responses.

Conclusion:
MyD88/CD40-based co-stimulation domains improve CAR-NK efficacy and persistence. Ongoing studies are evaluating downstream signaling, in vivo efficacy, and multi-omics to uncover mechanisms of enhanced functionality. These findings establish a foundation for safer, more durable CAR-NK therapies for cancer treatment.