CD86 costimulation enhances the antitumor activity of NKG2D CAR-Macrophages and synergizes with Anti-PD-L1 therapy to suppress prostate cancer progression.

Prostate cancer remains one of the most prevalent solid tumors in men worldwide and poses a major therapeutic challenge due to its immunologically "cold" tumor microenvironment (TME), which is characterized by low T cell infiltration, limited antigen presentation, and high levels of immunosuppressive factors. While chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematologic malignancies, it has yielded limited success in solid tumors such as prostate cancer. Macrophage-based CAR (CAR-M) therapies have emerged as promising alternatives, owing to macrophages' natural tumor-infiltrating capacity, phagocytic activity, and ability to modulate the TME. However, conventional CAR-Ms exhibit limited capacity to prime adaptive immunity due to insufficient co-stimulatory signaling. To address this limitation, we engineered two novel NKG2D-targeted CAR constructs for macrophages: one containing the intracellular Fc receptor common γ chain (FcRγ) alone (CAR-M), and another incorporating both FcRγ and the intracellular domain of the co-stimulatory molecule CD86 (CD86-CAR-M). These constructs were expressed in Raw264.7 macrophages or and evaluated for antigen-specific phagocytosis, tumor cell cytotoxicity, cytokine secretion, and T cell activation in vitro. In vivo efficacy and safety were assessed using both xenograft and syngeneic mouse models of prostate cancer, including combination therapy with anti-PD-L1 checkpoint blockade. Both CAR-M and CD86-CAR-M exhibited robust, antigen-specific phagocytosis and cytotoxicity against NKG2D ligand-expressing prostate cancer cells in vitro. CD86-CAR-Ms demonstrated enhanced M1 polarization, higher expression of CD86 and superior activation of cytotoxic T lymphocytes, accompanied by increased secretion of IFN-γ and TNF-α. This interaction triggers the phosphorylation of ERK, a central mediator of M1 macrophage polarization and inflammatory responses. In vivo, CD86-CAR-Ms achieved greater tumor suppression and survival benefit than CAR-Ms in immunocompetent models and significantly enhanced the efficacy of anti-PD-L1 antibody therapy without systemic toxicity. The improved anti-tumor effect was associated with increased T cell, NK cell infiltration and macrophages MHC-II expression, indicating TME reprogramming from cold to hot one. This study presents the first demonstration of CD86-enhanced CAR-macrophages targeting NKG2D ligands in prostate cancer. By integrating innate and adaptive immune activation, CD86-CAR-Ms represent a novel and potent strategy for overcoming immunosuppression and improving the therapeutic efficacy of immune checkpoint inhibitors in solid tumors.