Abstract A027: IL-12/IL-18 armored CAR-T cells drive pro-inflammatory remodeling of the glioblastoma tumor microenvironment
Authors: Shih Ryan M , Clubb Justin , Lam Katrina , Shafer Amanda , Vajragiri Shreya , Bouren Amber , Chen Yvonne 
Published: February 2025

Abstract
The standard of care for glioblastoma brain tumors has remained largely unchanged for 19 years. Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising therapeutic platform, but hallmark glioblastoma features—including a profoundly immunosuppressive and poorly immune-cell infiltrated tumor microenvironment (TME)—present significant challenges for engineered T cells. To address such challenges, CAR-T cells have increasingly been programmed to secrete “armors,” conferring additional functionality beyond tumor antigen binding. To that end, we engineered CAR-T cells targeting glioblastoma-associated antigen IL-13Rα2 to inducibly secrete pro-inflammatory cytokines IL-12 and IL-18 upon T-cell activation, and demonstrated robust anti-tumor efficacy against orthotopic glioma in immunocompetent mice. To deeply characterize the cellular composition of the glioma TME and the effects of CAR-T cell therapy on the TME, we analyzed brain-derived cells on days 2 and 9 post treatment (adoptive cell therapy [ACT]+2, ACT+9). At ACT+2, exogenous T cells were present in similar quantities in mice treated with armored and non-armored CAR-T cells, and endogenous T cells were comparatively rare. At ACT+9, however, CAR-12.18 treated mice exhibited profound expansion of both exogenous and endogenous T cells while mice receiving non-armored CAR-T cells exhibited contraction of those compartments. Immunophenotyping revealed expanded exogenous and endogenous T-cells in CAR-12.18–treated mice were differentially skewed in favor of CD8+ and CD4+ subtypes, respectively. Moreover, both populations exhibited increased expression of the resident memory marker CXCR6. These findings suggest formation of a multi-pronged adaptive immune response with capacity for long-term immunosurveillance. IL-12 and IL-18 armors also affected innate immune cell compartments. Excepting dendritic cells, there were no dramatic differences in myeloid or other immune cell populations in mice treated with armored and non-armored CAR-T cells at ACT+2. At ACT+9, however, CAR-12.18 treatment induced significant infiltration by both NK cells and monocytes, which was not observed in mice treated with non-armored CAR-T cells. These infiltration patterns suggest IL-12/IL-18 armors indirectly stimulated potent chemoattractive pathways. To track immune cell population dynamics with increased temporal resolution over a longer interval, we analyzed brain-derived cells from CAR-12.18–treated mice every three days from ACT+2 to ACT+17. Our results corroborated those from our previous ACT+2 and ACT+9 timepoints: the T-cell compartment underwent reconstitution from exogenous CD8+ cell dominance to endogenous CD4+ cell dominance, and we observed expansion and contraction phases of NK cell and monocyte recruitment. Altogether, these findings uncover widespread pro-inflammatory TME remodeling due to IL-12/IL-18 armors. We identify a CXCR6+ T-cell niche comprised of exogenous CD8 and endogenous CD4 T cells that, alongside NK cells and monocytes, may drive tumor regression and long-lasting immunity.
Citation Format: Ryan M Shih, Justin Clubb, Katrina Lam, Amanda Shafer, Shreya Vajragiri, Amber Bouren, Yvonne Chen. IL-12/IL-18 armored CAR-T cells drive pro-inflammatory remodeling of the glioblastoma tumor microenvironment [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A027.