Potent antitumor activity through dual targeting of PD-L1 and TGF-β pathways in the glioma tumor microenvironment

BACKGROUND: Glioblastoma, one of the most aggressive brain tumors, has been largely resistant to conventional immunotherapies, underscoring the need for novel treatment approaches. A promising strategy involves simultaneously inhibiting immunosuppressive pathways in the tumor microenvironment, as these pathways play pivotal roles in immune evasion. However, the therapeutic potential of combined targeting of these key immunosuppressive pathways in glioblastoma remains underexplored. We hypothesized that co-targeting the transforming growth factor (TGF)-β and PD-1 pathways could enhance immune responses against glioblastoma. METHODS: Human glioblastoma datasets were interrogated for the expression of PD-L1, TGF-β, and TGF-β target genes. Bintrafusp alfa, a first-in-class bifunctional fusion protein that blocks PD-L1 while sequestering TGF-β in the tumor microenvironment, was used to simultaneously inhibit both pathways. Its effects were assessed in vitro using human and mouse glioma cells and in vivo in immunocompetent, syngeneic mouse glioma models. High-dimensional flow cytometry was used to analyze treatment-induced changes in the tumor microenvironment. RESULTS: We observed a strong correlation between TGF-β and PD-L1 co-regulation, suggesting interconnected immunosuppressive mechanisms as part of a gene expression network. In vitro, bintrafusp alfa inhibited TGF-β-induced Smad2 phosphorylation, a bona fide response marker of TGF-β pathway activation, and enhanced immune cell-mediated killing of glioma cells. In vivo, combined targeting of both immunosuppressive pathways significantly improved survival of glioma-bearing mice, with long-term survivors exhibiting protection from tumor re-challenge. This survival benefit was not seen in T cell-deficient mice, confirming the necessity of adaptive immunity. High-dimensional flow cytometry of single-cell suspensions from tumor-bearing hemispheres revealed a distinct remodeling of immune subsets in the bintrafusp alfa-treated group compared with control-treated mice. CONCLUSIONS: Our findings provide strong support for the combined targeting of TGF-β and PD-L1 as a promising immunotherapeutic strategy to overcome immunosuppressive barriers in glioblastoma and induce potent antitumor responses.