Abstract
Glioblastoma is the most common lethal primary brain tumor, urging evaluation of new treatment options. Chimeric antigen receptor (CAR)-T cells targeting B7 homolog 3 (B7-H3) are promising because of the overexpression of B7-H3 on glioblastoma cells but not on healthy brain tissue. Nanobody-based (nano)CARs are gaining increasing attention as promising alternatives to classical single-chain variable fragment-based (scFv)CARs, because of their single-domain nature and low immunogenicity. Still, B7-H3 nanoCAR-T cells have not been extensively studied in glioblastoma. B7-H3 nanoCAR- and scFvCAR-T cells were developed and evaluated in human glioblastoma models. NanoCAR-T cells targeting an irrelevant antigen served as control. T cell activation, cytokine secretion and killing capacity were evaluated in vitro using ELISA, live cell imaging and flow cytometry. Antigen-specific killing was assessed by generating B7-H3 knock-out cells using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-genome editing. The tumor tracing capacity of the B7-H3 nanobody was first evaluated in vivo using nuclear imaging. Then, the therapeutic potential of the nanoCAR-T cells was evaluated in a xenograft glioblastoma model. We showed that B7-H3 nanoCAR-T cells were most efficient in lysing B7-H3glioblastoma cells in vitro. Lack of glioblastoma killing by control nanoCAR-T cells and lack of B7-H3glioblastoma killing by B7-H3 nanoCAR-T cells showed antigen-specificity. We showed in vivo tumor targeting capacity of the B7-H3 nanobody-used for the nanoCAR design-in nuclear imaging experiments. Evaluation of the nanoCAR-T cells in vivo showed tumor control in mice treated with B7-H3 nanoCAR-T cells in contrast to progressive disease in mice treated with control nanoCAR-T cells. However, we observed limiting toxicity in mice treated with B7-H3 nanoCAR-T cells and showed that the B7-H3 nanoCAR-T cells are activated even by low levels of mouse B7-H3 expression. B7-H3 nanoCAR-T cells showed promise for glioblastoma therapy following in vitro characterization, but limiting in vivo toxicity was observed. Off-tumor recognition of healthy mouse tissue by the cross-reactive B7-H3 nanoCAR-T cells was identified as a potential cause for this toxicity, warranting caution when using highly sensitive nanoCAR-T cells, recognizing the low-level expression of B7-H3 on healthy tissue.
Overview
- The study aims to evaluate the therapeutic potential of B7-H3 nanoCAR-T cells in glioblastoma therapy. The study developed and evaluated B7-H3 nanoCAR- and scFvCAR-T cells in human glioblastoma models. The study evaluated T cell activation, cytokine secretion, and killing capacity in vitro using ELISA, live cell imaging, and flow cytometry. The study assessed antigen-specific killing by generating B7-H3 knock-out cells using CRISPR/Cas9-genome editing. The study evaluated the tumor tracing capacity of the B7-H3 nanobody-used for the nanoCAR design in nuclear imaging experiments. The study evaluated the therapeutic potential of the nanoCAR-T cells in vivo in a xenograft glioblastoma model. The study showed that B7-H3 nanoCAR-T cells were most efficient in lysing B7-H3glioblastoma cells in vitro. The study showed in vivo tumor targeting capacity of the B7-H3 nanobody-used for the nanoCAR design in nuclear imaging experiments. The study showed tumor control in mice treated with B7-H3 nanoCAR-T cells in contrast to progressive disease in mice treated with control nanoCAR-T cells. However, the study observed limiting toxicity in mice treated with B7-H3 nanoCAR-T cells and showed that the B7-H3 nanoCAR-T cells are activated even by low levels of mouse B7-H3 expression. The study identified off-tumor recognition of healthy mouse tissue by the cross-reactive B7-H3 nanoCAR-T cells as a potential cause for this toxicity, warranting caution when using highly sensitive nanoCAR-T cells.
Comparative Analysis & Findings
- The study compared the outcomes observed under different experimental conditions or interventions detailed in the study. The study identified that B7-H3 nanoCAR-T cells were most efficient in lysing B7-H3glioblastoma cells in vitro. The study showed in vivo tumor targeting capacity of the B7-H3 nanobody-used for the nanoCAR design in nuclear imaging experiments. The study showed tumor control in mice treated with B7-H3 nanoCAR-T cells in contrast to progressive disease in mice treated with control nanoCAR-T cells. However, the study observed limiting toxicity in mice treated with B7-H3 nanoCAR-T cells and showed that the B7-H3 nanoCAR-T cells are activated even by low levels of mouse B7-H3 expression. The study identified off-tumor recognition of healthy mouse tissue by the cross-reactive B7-H3 nanoCAR-T cells as a potential cause for this toxicity, warranting caution when using highly sensitive nanoCAR-T cells.
Implications and Future Directions
- The study's findings suggest that B7-H3 nanoCAR-T cells have potential therapeutic value in glioblastoma therapy. However, the study identified limiting toxicity in mice treated with B7-H3 nanoCAR-T cells, which warrants caution when using highly sensitive nanoCAR-T cells. The study identified off-tumor recognition of healthy mouse tissue by the cross-reactive B7-H3 nanoCAR-T cells as a potential cause for this toxicity, which warrants further investigation. Future research could focus on developing nanoCAR-T cells with improved specificity for B7-H3glioblastoma cells and reduced toxicity. Future research could also investigate the use of nanoCAR-T cells in combination with other therapies, such as radiation or chemotherapy, to enhance their efficacy. Additionally, future research could investigate the use of nanoCAR-T cells in human glioblastoma models to further validate their therapeutic potential.