Deletion of Cbl-b inhibits CD8<sup>+</sup> T-cell exhaustion and promotes CAR T-cell function
Jitendra Kumar, Ritesh Kumar, Amir Kumar Singh, Elviche L. Tsakem, Mahesh Kathania, Matthew J. Riese, Arianne L. Theiss, Marco L. Davila, K. Venuprasad
Abstract
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.