Abstract A041: Vascular normalization improves the delivery and efficacy of CAR-T cells in mouse brain cancers
Authors: Salameti Vasiliki , Dong Xinyue , Ren Jun , Amoozgar Zohreh , Lee Somin , Datta Meenal , Roberge Sylvie , Duquette Mark , Fukumura Dai , Jain Rakesh K. 
Published: March 2024

Abstract
Introduction: Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematological malignancies, but its efficacy in patients with glioblastoma (GBM) and other solid tumors is limited due to poor tumor infiltration and an immunosuppressive tumor microenvironment (TME). We have previously shown that blocking vascular endothelial growth factor (VEGF) signaling can normalize tumor vessels, convert the immunosuppressive TME into an immunostimulatory environment, and enhance the efficacy of immunotherapy. This study aimed to investigate whether combining anti-VEGF therapy (B20) with EGFRvIII-targeting CAR-T cell treatment could improve delivery and effectiveness in immunocompetent orthotopic GBM mouse models. Methods: The study utilized two syngeneic mouse GBM cell lines (CT2A and GSC005) engineered to express EGFRvIII, a common neoantigen in human GBM. CAR-T cells were designed to target EGFRvIII. GBM-bearing mice were treated with B20[LS1] followed by EGFRvIII-CAR-T injection. Intravital imaging using two-photon microscopy was employed to track CAR-T cell infiltration into the tumor, while flow cytometry was used to measure the number and function of CAR-T and other immune cells. Results: The combination of B20 and CAR-T cell treatment significantly prolonged the survival of GBM-bearing mice compared to CAR-T cell therapy alone. Intravital imaging revealed that B20 normalized tumor vasculature, leading to a four-fold increase in the number of infiltrated CAR-T cells compared to CAR-T therapy without B20. Flow cytometry analysis showed an increased population of IL-2+ or IFN-γ+ CAR-T cells, indicating that B20 enhanced the anti-tumor function of injected CAR-T cells. Additionally, B20 therapy increased the population of endogenous lymphocytes, specifically Granzyme B+ TNF-α+ CD8 T cells (Cytotoxic T lymphocytes; CTLs) while decreasing FoxP3+ CD4 T cells (Regulatory T cells; Tregs). This indicated that the TME was remodeled to enhance the effect of CAR-T therapy. Conclusions: Our study demonstrated that combining B20 with CAR-T therapy improved their efficacy in GBM mouse models by enhancing CAR-T cell infiltration and reprogramming the TME to activate CAR-T cells and effector T cells. These findings provide compelling evidence for clinically evaluating the combination of anti-VEGF agents with CAR-T cells in GBM patients. We are now building on this approach to enhance CAR-T therapy for breast cancer brain metastasis. We are testing TNBC-targeting CAR-T therapy in murine TNBC-BrM[LS2] using a similar approach. Our hypothesis is that similar to the GBM results, anti-VEGF therapy and adoptive transfer of TNBC-targeting CAR-T cells [LS3] will improve CAR-T cell infiltration, delay tumor growth and prolong survival. This strategy has the potential to enhance emerging cellular immunotherapies' effectiveness in the brain tumor microenvironment.
Citation Format: Vasiliki Salameti, Xinyue Dong, Jun Ren, Zohreh Amoozgar, Somin Lee, Meenal Datta, Sylvie Roberge, Mark Duquette, Dai Fukumura, Rakesh K. Jain. Vascular normalization improves the delivery and efficacy of CAR-T cells in mouse brain cancers [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A041.