Journal Article

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

Kumar, Jitendra, Riese, Matthew J, Kumar Singh, Amir, Tsakem, Elviche L, Kumar, Ritesh, Theiss, Arianne L, Kathania, Mahesh, Davila, Marco L, Venuprasad, K

2021

Overview

BackgroundChimeric 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.MethodsAnalysis 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.ResultsOur 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.ConclusionOur 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.

• Publisher: BMJ Publishing Group LTD,BMJ Publishing Group
• Subject: Adaptor Proteins, Signal Transducing - genetics / Animals / Antigens / Cancer / Carcinoembryonic Antigen - metabolism / CD8-Positive T-Lymphocytes - immunology / Cloning / Colonic Neoplasms - genetics / Colonic Neoplasms - immunology / Colonic Neoplasms - therapy / Colorectal cancer / CRISPR / Enzymes / Experiments / Flow cytometry / Gene Expression Profiling / Gene Expression Regulation, Neoplastic / Humans / Immune Cell Therapies and Immune Cell Engineering / Immunotherapy / Immunotherapy, Adoptive / Interferon-gamma - metabolism / Kinases / Lymphocytes / Lymphocytes, Tumor-Infiltrating - immunology / Mice / Neoplasm Transplantation / Proto-Oncogene Proteins c-cbl - genetics / Receptors, Chimeric Antigen - metabolism / Sequence Analysis, RNA / Statistical analysis / Survival analysis / Tumor Microenvironment / Tumor Necrosis Factor-alpha - metabolism / Tumor necrosis factor-TNF / Tumors / Up-Regulation