PD1-TLR10 fusion protein enhances the antitumor efficacy of CAR-T cells in colon cancer

Background: The immunosuppressive microenvironment negatively affects the efficacy of chimeric antigen receptor T (CAR-T) cells in solid tumors. Fusion protein that combining extracellular domain of inhibitory checkpoint protein and the cytoplasmic domain of stimulatory molecule may improve the efficacy of CAR-T cells by reversing the suppressive signals.

Methods: To generate optimal PD1-TLR10 fusion proteins, PD1 extracellular domain and TLR10 intracellular domain were connected by transmembrane domain from PD1, CD28, or TLR10, respectively. The fusion protein was co-expressed with second generation anti-CEA CAR in the same retroviral vector. The effector function and the efficacy of fusion protein armored CAR-T cells was evaluated in vitro and in vivo.

Results: PD1-TLR10 armored CEA CAR-T cells showed stronger cytotoxicity and cytokine release against CEA-positive tumor cells. Specifically, CAR-T cells with fusion protein containing TLR10 transmembrane domain demonstrated better anti-tumor activity in xenograft mouse model.

Conclusion: Our study demonstrated that CEA CAR-T armored with rational designed PD1-TLR10 fusion protein had improved efficacy in colon cancer.