Membrane-immobilized gemcitabine for cancer-targetable NK cell surface engineering.

Although natural killer (NK) cell-based adoptive cell transfer (ACT) has shown promise in cancer immunotherapy, its efficacy against solid tumors is limited in the immunosuppressive tumor microenvironment (TME). Combinatorial therapies involving chemotherapeutic drugs such as gemcitabine (Gem) and NK cells have been developed to modulate the TME; however, their clinical application is constrained by low drug delivery efficiency and significant off-target toxicity. In this study, we developed cell membrane-immobilized Gem conjugates (i.e., lipid-Gem conjugates), designed to anchor seamlessly onto NK cell surfaces. Our modular-designed ex vivo cell surface engineeringmaterials comprise a lipid anchor for membrane immobilization, poly(ethylene glycol) to inhibit endocytosis, a disulfide bond as cleavable linker by glutathione (GSH) released during cancer cell lysis, and Gem for targeted sensitization. We demonstrated that the intrinsic properties of NK cells, such as proliferation and surface ligand availability, were preserved despite coating with lipid-Gem conjugates. Moreover, delivery of Gem prodrugs by lipid-Gem coated NK (GCNK) cells was shown to enhance antitumor efficacy against pancreatic cancer cells (PANC-1) through the following mechanisms: (1) NK cells recognized and attacked cancer cells, (2) intracellular GSH was leaked out from the lysed cancer cells, enabling cleavage of disulfide bond, (3) released Gem from the GCNK cells delivered to the target cells, (4) Gem upregulated MHC class I-related chain A and B on cancer cells, and (5) thereby activating NK cells led to enhance antitumor efficacy. The simultaneous co-delivery of membrane-immobilized Gem with NK cells could potentially facilitate both immune synapse-mediated cancer recognition and chemotherapeutic effects, offering a promising approach to enhance the anticancer efficacy of conventional ACTs.