Co-activating the intrinsic FcRγ/TLR4 signaling axis enhances the antitumor activity of NKG2D CAR-macrophages against prostate cancer.

Prostate cancer presents a major therapeutic challenge due to its immunosuppressive "cold" tumor microenvironment. To address this, we focused on harnessing the inherent phagocytic killing activity and immunomodulatory capacities of macrophages. Here, we explore an innovative strategy to enhance the therapeutic function of chimeric antigen receptor macrophages (CAR-Ms) through targeted engineering. We designed and constructed three distinct CAR-M variants targeting NKG2D ligands: a truncated control (Truncated-CAR-M), a construct containing the Fc receptor gamma chain (FcRγ) signaling domain (FcRγ-CAR-M), and a novel construct incorporating both the FcRγ and Toll-like receptor 4 (TLR4) intracellular domains (FcRγ-TLR4-CAR-M). In vitro, the FcRγ-TLR4-CAR-Ms demonstrated superior antigen-specific phagocytosis and tumor-killing activity, more stable pro-inflammatory M1 polarization, and enhanced T cell activation compared to other variants. Mechanistic studies revealed that these functional improvements were mediated through activation of the NF-κB signaling pathway. In an immunocompetent syngeneic mouse model of prostate cancer, treatment with FcRγ-TLR4-CAR-Ms resulted in significant tumor suppression and a survival benefit. This was associated with successful remodeling of the tumor immune microenvironment, and did not induce systemic toxicity. Our findings confirm that co-activating the intrinsic FcRγ/TLR4 signaling axis significantly enhances the anti-tumor efficacy and immunomodulatory capacity of CAR-Ms in prostate tumors.