Deletion of Cbl-b inhibits CD8 T-cell exhaustion and promotes CAR T-cell function.

BACKGROUND

Chimeric antigen receptor (CAR) T-cell therapy is an emerging option for cancer treatment, but its efficacy is limited, especially in solid tumors. This is partly because the CAR T cells become dysfunctional and exhausted in the tumor microenvironment. However, the key pathways responsible for impaired function of exhausted cells remain unclear, which is essential to overcome CAR T-cell exhaustion.

METHODS

Analysis of RNA-sequencing data from CD8 tumor-infiltrating lymphocytes (TILs) led to identification of Cbl-b as a potential target. The sequencing data were validated using a syngeneic MC38 colon cancer model. To analyze the in vivo role of Cbl-b in T-cell exhaustion, tumor growth, % PD1Tim3 cells, and expression of effector cytokines were analyzed in and mice. To evaluate the therapeutic potential of Cbl-b depletion, we generated a new CAR construct, hCEAscFv-CD28-CD3ζ.GFP, that recognizes human carcinoembryonic antigen (CEA). and CEA-CAR T cells were generated by retroviral transduction. mice bearing MC38-CEA cells were injected with and ; CEA-CAR T cells, tumor growth, % PD1Tim3 cells and expression of effector cytokines were analyzed.

RESULTS

Our results show that the E3 ubiquitin ligase Cbl-b is upregulated in exhausted (PD1Tim3) CD8 TILs. CRISPR-Cas9-mediated inhibition of Cbl-b restores the effector function of exhausted CD8 TILs. Importantly, the reduced growth of syngeneic MC38 tumors in mice was associated with a marked reduction of PD1Tim3 CD8 TILs. Depletion of Cbl-b inhibited CAR T-cell exhaustion, resulting in reduced MC38-CEA tumor growth, reduced PD1Tim3 cells and increased expression of interferon gamma, tumor necrosis factor alpha, and increased tumor cell killing.

CONCLUSION

Our studies demonstrate that deficiency of Cbl-b overcomes endogenous CD8 T-cell exhaustion, and deletion of Cbl-b in CAR T cells renders them resistant to exhaustion. Our results could facilitate the development of efficient CAR T-cell therapy for solid tumors by targeting Cbl-b.