Engineered bioorthogonal cell delivery system for in situ antimicrobial peptide recruitment during systemic bacterial...

Allogeneic cells represent promising intelligent delivery platforms owing to their intrinsic target homing ability, ready-to-use, scalability and broad applicability. However, implanted allogeneic cells are susceptible to rapid clearance by mononuclear phagocytic system (MPS), resulting in short half-life and compromised therapeutic efficacy. To overcome this limitation, we constructed genetically engineered allogeneic cells with surface-expressed CD24, a "don't eat me" signal protein, to evade phagocytosis by macrophages. Additionally, we modified the allogeneic cells with azide groups, creating a binding site for dibenzocyclooctyne (DBCO)-modified drugs through copper-free click chemistry. The results showed that mesenchymal stem cells (MSCs) have natural inflammation-targeting properties, and modification of allogeneic MSCs (M24N₃ cells) significantly prolonged their retention at the site of inflammation. Moreover, DBCO-modified antimicrobial peptides (DBCO-LL37) were more effectively recruited to inflammation sites via bioorthogonal reactions, resulting in sustained bacterial clearance. The M24N₃@DBCO-LL37 treatment cleared multiple sepsis mediators, extended circulation time, and increased tissue retention, ultimately protecting against organ damage and delaying sepsis-induced lethality, subsequently resulting in remarkable survival rate elevation. These findings underscore the potential of bioorthogonal system based on engineered allogeneic cells for the treatment of complex inflammatory diseases, highlighting their promising applications in evading rapid clearance systems in vivo. STATEMENT OF SIGNIFICANCE: In recent years, allogeneic cells have garnered significant research interest as emerging drug delivery carriers due to their off-the-shelf availability, scalable production, and broad therapeutic applicability. However, recognition and elimination mediated by the mononuclear phagocyte system (MPS) brings a substantial challenge to their clinical application. We developed an engineered bioorthogonal cell delivery system, M24N₃@DBCO-LL37, through genetic engineering and glucose metabolic engineering methods, which could avoid phagocytosis of allogeneic cells by macrophages, prolong the retention time of allogeneic cells at the site of inflammation, recruit more DBCO-modified antimicrobial peptides (DBCO-LL37), and significantly reduced the mortality rate and improved therapeutic efficiency in a mouse model of sepsis. This strategy can not only be used in the development of cell delivery systems, but also has the potential to be used in the design of more allogeneic cell therapy strategies, such as chimeric antigen receptor T-cell immunotherapy (CAR-T), haematopoietic stem cell transplantation and organ transplantation, to improve the therapeutic efficacy.