Targeting hypoxia downstream signaling protein, CAIX, for CAR T-cell therapy against glioblastoma

Abstract

Background Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to (i) develop chimeric antigen receptor (CAR) T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and (ii) demonstrate its efficacy with limited off-target effects. Methods First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR T cells were generated against CAIX and efficacy was assessed in 4 glioblastoma cell lines and 2 glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR T cells was assessed via interferon gamma, tumor necrosis factor alpha, and interleukin-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intratumoral injection of the anti-CAIX CAR T cells on an in vivo xenograft mouse model using the U251 luciferase cell line. Tumor infiltrating lymphocyte analyses were performed. Results We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR T-cell therapy using anti-CAIX CAR T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects. Conclusions By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR T-cell therapy, our data suggest that anti-CAIX CAR T may be a promising strategy to treat glioblastoma. Direct intratumoral injection increases anti-CAIX CAR T-cell potency while limiting its off-target effects.

Inhibition of PP2A with LB-100 Enhances Efficacy of CAR-T Cell Therapy Against Glioblastoma

“…To investigate if LB-100 similarly enhanced the cytotoxicity profile of the anti-CAIX CAR-T cell, we performed a series of in vitro experiments using four glioblastoma cell lines (T98G, A172, LN229, and U251). As we previously reported, U251 demonstrated more sensitivity to hypoxia-induced CAIX expression when compared to the other three cell lines [14]. Here, we also evaluated the U251-Luc glioblastoma cell line, which can be used to generate an orthotopic xenograft mouse model for in vivo study.…”

“…To assess in vitro functionality of the anti-CAIX CAR-T cells combined with LB-100 against glioblastoma cells, tumor cells were co-cultured with anti-CAIX CAR-T cells or control T cells and treated with titration of LB-100 for 48 h. As we previously demonstrated, anti-CAIX CAR-T cells showed dose-dependent cytotoxicity on U251 cells in different effector (E):target (T) ratios, ranging from 1:5 to 5:1 [14]. Higher E:T ratio showed more significant cytotoxicity.…”

“…We first investigated the impact of LB-100 on CAR-T cells alone. Anti-CAIX CAR-T cells were generated as previously reported [14]. The transduction efficiency was evaluated using a mock vector (containing a green fluorescent protein-GFP expression element instead of CAIX scFv sequences) transduced T cells and assessed by GFP expression 4 days post-transduction.…”

“…Mice were similarly implanted with U251-Luc tumors and randomized into the following four treatment groups: Un-treated, LB-100, anti-CAIX CAR-T, and Combo (LB-100 plus anti-CAIX CAR-T). After two weeks of treatment, brain tumors were harvested and analyzed by flow cytometry with human T cell markers (CD3 + , CD4 + , and CD8 + ) according to the previously described gating strategy [14].…”

“…In particular, the EGFRvIII, IL13Rα2, HER2-targeted CAR-T therapy demonstrated promising results in the clinical 2 of 15 setting [5,[10][11][12][13]. In addition, we previously showed that CAR-T cells targeting Carbonic anhydrase IX (CAIX), a membrane-bound protein involved in maintaining intracellular pH that is upregulated in glioblastoma tumor cells, curtailed tumor growth and improved survival in glioblastoma-bearing mice [14,15].…”