Synergistic antitumor activity of CAR-NK cells combined with CAR-macrophages for pancreatic cancer treatment.

[BACKGROUND] Adoptive cell therapies employing chimeric antigen receptors (CARs) have achieved remarkable success in hematologic malignancies, yet their efficacy in solid tumors remains limited due to the immunosuppressive tumor microenvironment (TME), which restricts immune cell infiltration and persistence. While CAR-NK cells provide potent cytotoxicity with a favorable safety profile, their tumor penetration is often suboptimal. In contrast, macrophages possess intrinsic tumor-homing and tissue-remodeling abilities that could help overcome these barriers.

[OBJECTIVE] This study aimed to develop a combinatorial innate-cell immunotherapy integrating nanobody-based mesothelin (MSLN)-targeting CAR-macrophages (CAR-Ms) with CAR-NK cells to remodel the TME and enhance antitumor immunity in pancreatic cancer.

[METHODS] Using a validated D3 nanobody CAR construct, CAR-Ms were generated from PMA-differentiated THP-1 monocytes and characterized for CAR expression, M1/M2 polarization, migration, phagocytosis, and cytokine secretion. CAR-NK cell migration and cytotoxicity were evaluated using conditioned media (CM) from CAR-M/tumor co-cultures. Synergistic antitumor activity was assessed in an orthotopic MSLN⁺ pancreatic ductal adenocarcinoma (PDAC) model.

[RESULTS] D3-CAR-Ms exhibited robust CAR expression (> 90%) and sustained an M1-like phenotype (CD86⁺HLA-DR⁺CD204⁻) even after tumor engagement. Functionally, they displayed enhanced migration and infiltration into MSLN⁺ PANC-1 spheroids, along with increased phagocytic and tumoricidal activity. Upon antigen engagement, CAR-Ms secreted high levels of CXCL9, which promoted CAR-NK chemotaxis, degranulation, and cytotoxicity. Sequential administration of CAR-Ms and CAR-NK cells in vivo led to marked tumor regression and durable responses without systemic toxicity.

[CONCLUSION] Combinatorial nanobody-based CAR-M and CAR-NK therapy reprograms the TME and establishes a CXCL9-driven feed-forward loop between macrophages and NK cells, leading to synergistic innate immune activation and potent tumor control. This strategy provides a mechanistically grounded and translationally feasible framework for next-generation CAR-based immunotherapies targeting MSLN-expressing solid tumors.