Extracellular Vesicles Derived from Natural Killer Cells as a Therapeutic Strategy for Triple-Negative Breast Cancer

Abstract

Natural killer cell-derived extracellular vesicles (NK-EVs) represent a promising new dual-class of cancer immunotherapeutics, combining the intrinsic cytotoxicity of NK cells and their immunomodulatory function with the advantages of a cell-free nanovesicle system. Their small size, stability, tumour tropism, and ability to cross biological barriers position them as attractive candidates for treating hard-to-treat malignancies, such as triple-negative breast cancer (TNBC). However, their translation into clinical settings requires establishing scalable manufacturing methods, validating potency assays, and deepening the mechanistic understanding of their immunomodulatory activity. This thesis, composed of three central studies, addresses these challenges. In Study 1, a Good Manufacturing Practice-compliant biomanufacturing workflow was developed using a closed-loop, perfusion-powered hollow-fibre bioreactor system to produce NK-EVs from the clinically relevant NK92-MI cell line continuously. Under serum-free, xeno-free, and feeder-free conditions, this process generated large quantities of viable NK cells and clinical-grade NK-EVs while retaining cytotoxic effectors and pro-inflammatory cytokines. The resulting products exhibited potent cytotoxicity against leukemic cells with minimal off-target toxicity against healthy cells, providing a scalable platform for clinical-grade NK-EV production. In Study 2, a highly sensitive resazurin phenoxazine-based viability assay was validated as a potency assay to assess the cytotoxic function of NK-EVs. The assay reliably quantified NK-EV-mediated cytotoxicity against both suspension- and adherent-cancer models, demonstrating specificity & sensitivity, linearity & range, reproducibility, and stability. Importantly, NK-EV potency correlated with effector proteins and detected loss of function in degraded samples, establishing it as a robust analytical method for biotherapeutic development. In Study 3, the immunomodulatory capacity of NK-EVs was investigated using high-dimensional flow cytometry, single-cell RNA sequencing and functional assays. NK-EVs activated and reprogrammed immune subsets, including cytotoxic CD8+ T cells and NK cells, enhancing coordinated anti-cancer responses in both healthy donors and breast cancer patients. Collectively, this work establishes the foundation for translating NK-EVs into a novel cell-free immunotherapy for hard-to-treat cancers.