Chimeric antigen receptor (CAR) T cells have shown limited efficacy against solid tumors because of poor tissue penetration, constrained activity, and early exhaustion due to the immunosuppressive tumor microenvironment (TME). Although stimulatory cytokines can counteract immune suppression, their systemic administration entails risk of toxicities and counter-regulatory responses. Here, we leveraged a population of tumor-associated TIE2-expressing macrophages (TEMs) to release interferon-α (IFN-α) and/or orthogonal interleukin-2 (oIL2) at the tumor site. Targeted cytokine delivery rescued CAR T cell functionality against the clinically relevant tumor antigen B7-homolog 3 (B7-H3) in an orthotopic, CAR T cell–refractory, immunocompetent mouse model of glioblastoma (GBM) named mGB2 that recapitulates pathological features of the human disease. Immunophenotypic and transcriptomic analyses revealed that inhibition of premature terminal exhaustion and induction of effector and memory states featuring activation of signaling pathways and transcriptional networks putatively boosted CAR T cell antitumor activity. Furthermore, IFN-α, especially when combined with private oIL2 signaling to CAR T cells, elicited potent endogenous T cell responses against multiple tumor-associated antigens, leading to delayed GBM growth and prolonged mouse survival even with tumors expressing B7-H3 in only a fraction of cells. These data suggest that the combination of TEM-based cytokine delivery and CAR T cells may have synergistic effects and support the further study of this approach for the treatment of patients with GBM.

Among Histone post-translational modifications (PTMs), lysine acetylation plays a pivotal role in the epigenetic regulation of gene expression, mediated by chromatin modifying enzymes. Due to their activity in physiology and pathology, several chemical compounds have been developed to inhibit the function of these proteins. However, the pleiotropy of these classes of proteins represents a weakness of epigenetic drugs. Ideally, a new generation of epigenetic drugs should target with molecular precision individual acetylated lysines on the target protein. We exploit a PTM-directed interference, based on an intrabody (scFv-58F) that selectively binds acetylated lysine 9 of histone H3 (H3K9ac), to test the hypothesis that targeting H3K9ac yields more specific effects than inhibiting the corresponding HAT enzyme that installs that PTM. In yeast scFv-58F modulates, gene expression in a more specific way, compared to two well-established HAT inhibitors. This PTM-specific interference modulated expression of genes involved in ribosome biogenesis and function. In mammalian cells, the scFv-58F induces exclusive changes in the H3K9ac-dependent expression of specific genes. These results suggest the H3K9ac-specific intrabody as the founder of a new class of molecules to directly target histone PTMs, inverting the paradigm from inhibiting the writer enzyme to acting on the PTM.