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, % PD1 + Tim3 + cells, and expression of effector cytokines were analyzed in cbl-b +/+ and cbl-b -/- 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). cbl-b +/+ and cbl-b -/- CEA-CAR T cells were generated by retroviral transduction. Rag -/- mice bearing MC38-CEA cells were injected with cbl-b +/+ and cbl-b -/-; CEA-CAR T cells, tumor growth, % PD1 + Tim3 + cells and expression of effector cytokines were analyzed. Results: Our results show that the E3 ubiquitin ligase Cbl-b is upregulated in exhausted (PD1 + Tim3 +) 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 cbl-b -/- mice was associated with a marked reduction of PD1 + Tim3 + CD8 + TILs. Depletion of Cbl-b inhibited CAR T-cell exhaustion, resulting in reduced MC38-CEA tumor growth, reduced PD1 + Tim3 + 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.