Background CAR-based cell therapies are hampered by a lack of cell surface target proteins that are also good tumor-specific antigens. HLA-G is an exception as it is only highly expressed in cytotrophoblasts of the human placenta where it protects the fetus from all arms of the immune system with very little expression elsewhere. HLA-G directs immunosuppression by binding to ILT receptors (ILT2 in lymphoid cells and ILT4 in myeloid cells) via ITIM signaling domains. HLA-G is derepressed in about 50% of solid tumors and leukemias, notably Acute Myeloid Leukemias (AML).
Methods CD56+ NK cells were selected from PBMCs, incubated with IL-15 and activated with a feeder free cocktail of an immobilized cytokine and activating ligand. Activated NK cells were transduced with γ-retroviruses directing expression of CIR proteins, soluble IL-15 and a ΔCD19 marker protein or CAR control constructs directed to HLA-G or CD33. After 8 days of expansion, CIR- or CAR-NK cells were cocultured with human GFPffluc-expressing AML target cell lines with or without transgenic HLA-G G1, G2 and G5 isoforms. Cytotoxicity of CIR-NK cells was determined by reduction of GFP fluorescence or luciferase activity. In comparative experiments, T cells from PBMCs were transduced to produce CIR-T cells by standard methods.
Results T and NK cells were efficiently and stably transduced by the CIR constructs. In cocultures with THP-1 AML cells (HLA-G negative), mock-transduced NK cells displayed innate killing activity that was not augmented by CIR constructs. In cocultures against THP-1-GFPffluc target cells expressing exogenous G1, G2 or G5 isoforms, CIR-NK cells or CIR-T cells displayed augmented target cell killing in 2-day and 7-day assays relative to mock-transduced effector cells (figure 1). CIR constructs with the two N-terminal domains showed enhanced HLA-G targeting. ILT4 CIR-T cells recognized target cells expressing HLA-G2 that were not recognized by HLA-G CAR-T cells. Further, AML cell lines MOLM13 and Kasumi1 express low endogenous levels of HLA-G1 and G5. In cocultures, CIR-NK cells displayed potent serial killing activity of both cell lines accompanied by secretion of IFN-γ at E:T ratios of 1:10 and 1:20. This anti-tumor efficacy and IFN-γ production were further enhanced in a screen for potent coactivation domains other than 4–1BB that activate innate immunity.
Conclusions This technology takes advantage of the physiological pairing of the ILT receptors with the multiple immunosuppressive HLA-G isoforms. Engineered CIR-NK cells can convert HLA-G expression in AML from a potent inhibitor of lymphocyte activity into a target for anti-tumor control.
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