CAR microglia-like anti‑carbonic anhydrase IX releasing anti-programmed cell death ligand-1 antibodies for...

Glioblastoma (GBM) is characterized by profound immunosuppression and limited responsiveness to conventional T cell-based therapies and immune checkpoint blockade. Here, we establish a human microglial cell line to explore the concept of a brain-adapted platform for chimeric antigen receptor (CAR) expression targeting GBM. We engineered human microglial cells with anti‑carbonic anhydrase IX (CAIX) CARs incorporating either CD28 or 4-1BB costimulatory domains, alone or co-expressing anti-programmed cell death ligand 1 (PD-L1) monoclonal antibodies (mAbs). Our results demonstrate that CAIX-targeted CAR-engineered microglia-like cells (CAR-MG) mediate potent, antigen-dependent tumor killing in vitro, with CD28-based constructs exhibiting superior antitumor efficacy and a robust pro-inflammatory cytokine profile. Furthermore, local secretion of anti-PD-L1 synergized with CAIX-targeting to significantly reduce tumor burden in vivo - an effect closely associated with the successful reprogramming of infiltrating myeloid cells toward an M1-like anti-tumoral state, evidenced by an increase in the frequency of cells expressing CD38, CD86, and HLA-DR. Notably, the anti-PD-L1 secretory platform downregulated tumor-derived PD-L1, effectively remodeling the immunosuppressive glioblastoma microenvironment. These findings suggest that this engineered microglial cell line platform has the potential to heat the immunologically "cold" GBM microenvironment. Together, these results identify CAIX-targeted engineered CAR-MG as an effective immunotherapeutic approach and support further development of CNS-targeted immune cells to overcome key barriers to cellular immunotherapy in GBM.