Anti-VEGF therapy improves EGFR-vIII-CAR-T cell delivery and efficacy in syngeneic glioblastoma models in mice

Abstract

Chimeric antigen receptor (CAR)-T cells have revolutionized the treatment of multiple types of hematological malignancies, but have shown limited efficacy in patients with glioblastoma (GBM) or other solid tumors. This may be largely due to the immunosuppressive tumor microenvironment (TME) that compromises CAR-T cells' delivery and antitumor activity. We previously showed that blocking vascular endothelial growth factor (VEGF) signaling can normalize tumor vessels in murine and human tumors, including GBM, breast, liver, and rectal carcinomas. Moreover, we demonstrated that vascular normalization can improve the delivery of CD8+ T cells and the efficacy of immunotherapy in breast cancer models in mice. In fact, the US FDA (Food and drug administration) has approved seven different combinations of anti-VEGF drugs and immune checkpoint blockers for liver, kidney, lung and endometrial cancers in the past 3 years. Here, we tested the hypothesis that anti-VEGF therapy can improve the delivery and efficacy of CAR-T cells in immunocompetent mice bearing orthotopic GBM tumors. We engineered two syngeneic mouse GBM cell lines (CT2A and GSC005) to express EGFRvIII-one of the most common neoantigens in human GBM-and CAR T cells to recognize EGFRvIII. We found that treatment with the anti-mouse VEGF antibody (B20) improved CAR-T cell infiltration and distribution throughout the GBM TME, delayed tumor growth, and prolonged survival of GBM-bearing mice compared with EGFRvIII-CAR-T cell therapy alone. Our findings provide compelling data and a rationale for clinical evaluation of anti-VEGF agents with CAR T cells for GBM patients.

Keywords: tumor microenvironment.

Figure 1

Anti-VEGF treatment improves the efficacy of EGFRvIII-CAR-T cells in murine GBM tumor models. (A) Schematic representation of experimental setup to evaluate the effect of PBS, CAR-T, B20, IgG+CAR T and B20+CAR T on the survival of GSC005 and C2TA GBM-bearing mice. (B) and (C) Tumor growth kinetics and median survival for CT2A tumors (PBS (n=22, 15.5 days), CAR-T (n=14, 20.5 days), B20 (n=8, 24.5 days), B20+CAR T (n=19, 32 days)). (D) and (E) Tumor growth kinetics and median survival for GSC005 tumors (PBS (n=12, 13.5 days), CAR-T (n=17, 18.5 days), B20 (n=10, 24 days), IgG+CAR T (n=13, 18 days), B20+CAR T (n=21, 37 days)). Error bars show median±SEM. Statistical analysis was performed using Student’s t-test or one-way ANOVA test. *p<0.05, ****p<0.0001. ANOVA, analysis of variance; CAR, chimeric antigen receptor; GBM, glioblastoma; VEGF, vascular endothelial growth factor. PBS, phophate buffered saline.

Figure 2

Anti-VEGF treatment improves the infiltration of EGFRvIII-CAR-T cells. (A) Intratumoral distribution of EGFRvIII-CAR-T cells inside of GFP-GSC005 GBM tumors imaged by multiphoton microscopy. Images were taken 24 hours after the injection of CAR-T cells (fluorescently labeled as red). Scale bars=50 µm. (B) Measurement of CAR-T cell (pink) number in GFP-GSC005 tumors (green) treated with PBS or B20 (2.5 mg/kg). scale bar=50 µm. (C) Percentage of EGFRvIII-CAR-T cells inside of GSC005 GBM tumors measuring by flow cytometry. Error bars show ±SEM. Statistical analysis was performed using Student’s t-test. *p<0.05, **p<0.01. CAR, chimeric antigen receptor; GBM, glioblastoma. PBS, phosphate buffered saline.

Figure 3

Anti-VEGF treatment enhances function of CAR-T cells and effector T cells in GBM. (A) GSC005 tumors were treated as shown in the schematic. (B) Percentage of IL2+and IFN-γ+ CAR T cells inside the GSC005 GBM tumors. (C) Percentage of CD4+T cells, CD8+T cells, and the cytotoxic Granzyme B+TNF-α+ CD8+ T cells in the GSC005 tumors. Error bars show ±SEM. Statistical analysis was performed using Student’s t-test. *p<0.05, **p<0.01, ***p<0.001. CAR, chimeric antigen receptor; VEGF, vascular endothelial growth factor.