Category: Preclinical Development
Purpose: Chimeric antigen receptors (CAR)-engineered T cell therapies have achieved remarkable success in hematological malignancies; however, in solid tumor treatment, CAR T therapies have not shown the same level of clinical benefit due to multiple factors. In our previous study, we engineered masked anti-EGFR CAR T cells that could be activated by tumor-associated protease to mitigate “on-target off-tumor” toxicity observed in CAR T therapies targeting solid tumor antigen. Despite promising in vitro results, the masked anti-EGFR CAR T therapy, like many other CAR T therapies targeting solid tumor antigens, could only convey limited antitumor efficacy in xenograft model due to attenuated persistence of the CAR T cells. In this scenario, the CAR T cells circulate in blood without antigen stimulation before they get activated at tumor site, which is unfavorable for CAR T cell persistence and expansion. To overcome this limitation, we designed a novel tandem CAR construct by linking masked anti-EGFR scFv with anti-CD19 scFv in the extracellular region, aiming to provide CAR T cells with stimulation signal by B cells in blood.
Methods: To evaluate the function the tandem CAR T cells in vitro, we studied the expression of IFN-γ, antigen specific cell lysis, and T cell expansion upon antigen stimulation following antigen-specific stimulation. Furthermore, the antitumor efficacy of the tandem CAR T cells combined with CD19 positive cells that mimic B cells was determined using a xenograft mouse model.
Results: By preparing retroviral vectors by transient transfection of 293T cells and transducing activated human PBMC cells, we successfully generated the masked EGFR-CD19 CAR T cells. In vitro studies have demonstrated that, like masked EGFR CAR T cells, masked EGFR-CD19 CAR T cells could bind to and get stimulated by EGFR in the presence of tumor associated protease, while they could also get stimulation signal by recognizing CD19+ target cells. Co-culture with CD19+ target cells promoted the expression of the functional marker IFN-gamma and the proliferation marker Ki67. In in vivo studies, we utilized irradiated K562-CD19 cells to mimic human B cells in body. The co-administration of the masked EGFR-CD19 CAR T cells and K562-CD19 cells showed significantly enhanced antitumor efficacy in NCI-H292 xenograft mouse model compared with masked EGFR CAR T cells or masked EGFR-CD19 CAR T cells, or masked EGFR CAR T cells combined with K562-CD19 cells. Further ex vivo data indicated that K562-CD19 cells improved the persistence of the masked EGFR-CD19 CAR T cells, which could be beneficial for the masked EGFR-CD19 CAR T cells to eradicate solid tumor.
Conclusion: With the stimulation from B cells, the tandem CAR T cells that can be stimulated by both CD19 and tumor associated antigen exhibit enhanced antitumor efficacy and persistence.
Pin Wang– Los Angeles, California