One of the main challenges for the translation of CAR-T cell-based therapies to the treatment of solid tumors is the identification of specific antigens on the tumor cell membrane. In this work, we focused on phosphatidylserine (PS), a phospholipid normally present on the inner surface of the cell membrane, which is externalized during apoptosis acting as an “eat-me” signal for macrophages allowing their silent removal. PS externalization is highly deregulated in cancer cells and tumor microenvironment, and, thus, it could be considered as a tumor antigen for CAR-T cell designs based on AnnexinV, a protein with high affinity for PS.

We demonstrated that PS is widely exposed in many tumor cell lines and tumor tissues. AnxaV CAR-T cells recognized PS on the surface of tumor cells. However, PS is also exposed in T lymphocytes after activation, which triggered a fratricide effect in AnxaV CAR-T cells. This could be bypassed by using an inducible AnxaV CAR. As an alternative, we prepared the bi-functional protein EDA-AnxaV to redirect the antigen specificity of EDA CAR-T cells to PS-expressing tumors. We found that EDA CAR-T cells can kill PS-expressing tumor cells in the presence of the adaptor EDA-AnxaV in vitro, showing a trend to improve the antitumor activity of EDA CAR-T therapy in vivo in the F9 teratocarcinoma tumor model.

In conclusion, PS could be considered as a potential tumor antigen for CAR-T cell-based therapies when the fratricide effect on CAR-T cells is bypassed by inducible CAR constructs or by using bi-functional adaptor proteins.