Engineering CAR-NK Cells with Enhanced Persistence and Functionality for Cancer Immunotherapy

Abstract

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of hematologic malignancies, but its application remains limited by manufacturing constraints, toxicity, and antigen escape. CAR-engineered natural killer (CAR-NK) cells represent a promising alternative due to their favorable safety profile, reduced risk of graft-versus-host disease, and capability for off-the-shelf use. However, a main limitation of CAR-NK therapy is poor in vivo persistence, largely due to dependence on exogenous cytokine support. This study addresses this challenge by developing a targeted cytokine delivery strategy, the CAR-Enhancer (CAR-E) platform on NK cells. CAR-Es are bifunctional molecules composed of a CAR-targeting antigen domain fused to a low-affinity mutant cytokine, enabling selective delivery of cytokine signaling exclusively to CAR-expressing NK cells. This design concentrates survival and activation signals at the CAR interface while minimizing systemic toxicity and off-target immune activation. First, we demonstrate that IL-2–based CAR-E constructs selectively bind CAR-NK cells and induce potent STAT5 signaling in a CAR-dependent manner, while sparing non-transduced immune cells. This targeted signaling results in preferential expansion and functional activation of CAR-NK cells. Functionally, CAR-E enhances CAR-NK cytotoxicity against tumor targets without impairing CAR-antigen engagement. Notably, CAR-E also boosts NK-intrinsic cytotoxic pathways, enabling enhanced tumor killing even in the absence of canonical CAR signaling. This dual mechanism demonstrates a broader immunomodulatory role for CAR-E, beyond traditional CAR-mediated targeting. In vivo, exogenous CAR-E administration promotes robust CAR-NK cell expansion, multi-organ persistence, and complete tumor control in a xenograft model of multiple myeloma. Persisting CAR-NK cells retain an activated, non-exhausted phenotype and preserved cytotoxic function, demonstrating durable functional competence. To further improve translational feasibility, we engineered armored CAR-NK cells that autonomously secrete CAR-E via a bicistronic T2A construct. This self-secretion system increases cytotoxicity, metabolic fitness, and resistance to exhaustion under chronic tumor exposure. In vivo, armored CAR-NK cells demonstrate prolonged persistence and improved tumor control even when tumor challenge is delayed, showing their potential for long-term immunosurveillance. This work confirms CAR-E as a novel and versatile strategy to enhance CAR-NK cell persistence and functionality. By enabling targeted cytokine delivery without systemic toxicity, the CAR-E platform addresses a central limitation of CAR-NK therapy and provides a broadly applicable framework for improving adoptive cell therapies in cancer immunotherapy.