Binding and Efficacy of Anti-Robo4 CAR-T Cells against Solid Tumors

Chimeric antigen receptor expression T (CAR-T) cell therapy has been shown be efficacious against relapsed/refractory B-cell malignant lymphoma and has attracted attention as an innovative cancer treatment. However, cells of solid tumors are less accessible to CAR-T cells; moreover, CAR-T function is decreased in the immunosuppressive state of the tumor microenvironment. Since most tumors induce angiogenesis, we constructed CAR-T cells targeting roundabout homolog 4 (Robo4), which is expressed at high levels in tumor vascular endothelial cells, by incorporating three anti-Robo4 single-chain variable fragments (scFv) that were identified using phage display. We found that binding affinities of the three CARs to mouse and human Robo4 reflected their scFv affinities. More importantly, when each CAR-T cell was assayed in vitro, antigen-specific cytotoxicity, cytokine-producing ability, and proliferation were correlated with binding affinity for Robo4. In vivo, all three T-cells inhibited tumor growth in a B16BL6 murine model, which also correlated with Robo4 binding affinities. However, growth inhibition of mouse Robo4-expressing tumors was observed only in the model with CAR-T cells with the lowest Robo4 affinity. Therefore, at high Robo4 expression, CAR-T in vitro and in vivo were no longer correlated, suggesting that clinical tumors will require Robo4 expression assays.