Targeting of the CD161 Inhibitory Receptor Enhances T cell-mediated Immunity Against Hematological Malignancies

The CD161 inhibitory receptor is highly upregulated by tumor-infiltrating T cells in a number of human solid tumor types, and its ligand CLEC2D is expressed by both tumor cells and infiltrating myeloid cells. Here we assessed the role of the CD161 receptor in hematological malignancies. Systematic analysis of CLEC2D expression using the Cancer Cell Line Encyclopedia (CCLE) revealed that CLEC2D mRNA was most abundant in hematological malignancies, including B cell and T cell lymphomas as well as lymphocytic and myelogenous leukemias. CLEC2D protein was detected by flow cytometry on a panel of cell lines representing a diverse set of hematological malignancies. We therefore used yeast display to generate a panel of high-affinity, fully human CD161 monoclonal antibodies (mAbs) that blocked CLEC2D binding. These mAbs were specific for CD161 and had a similar affinity for human and non-human primate CD161, a property relevant for clinical translation. A high-affinity CD161 mAb enhanced key aspects of T cell function, including cytotoxicity, cytokine production and proliferation, against cell lines originating from patients with AML, DLBCL and Burkitt lymphoma. In humanized mouse models, this CD161 mAb enhanced T cell-mediated immunity resulting in a significant survival benefit. ScRNA-seq data demonstrated that CD161 mAb treatment enhanced expression of cytotoxicity genes by CD4 T cells as well as a tissue-resident memory program by CD4 and CD8 T cells that is associated with favorable survival outcomes in multiple human cancer types. These fully human monoclonal antibodies thus represent potential immunotherapy agents for hematological malignancies. Overall design: NSG mice were injected I.V. with NY-ESO-1+ Raji cells, followed by I.V. injection of NY-ESO-1 TCR+ CD161+ cells. Mice were treated with either isotype control or CD161 blocking antibody (n=5 per treatment group). On day 13, cells were isolated from both hind limbs and FACS sorted to isolate live CD3+ T cells (human CD45+ human CD19neg, ZsGreenneg, mouse CD45neg). Cells were analyzed by scRNA-seq using the 10X Genomics platform.