Effects of anti-NKG2A antibody administration on leukemia and normal hematopoietic cells

Abstract

Natural killer cells are key cells of the innate immune system. Natural killer cell receptor repertoires are diversified by a stochastic expression of killer-cell-immunoglobulin-like receptors and lectin-like receptors such as NKG2 receptors. All individuals harbor a subset of natural killer cells expressing NKG2A, the inhibitory checkpoint receptor for HLA-E. Most neoplastic and normal hematopoietic cells express HLA-E, the inhibitory ligand of NKG2A. A novel anti-human NKG2A antibody induced tumor cell death, suggesting that the antibody could be useful in the treatment of cancers expressing HLA-E. We found that immunodeficient mice, co-infused with human primary leukemia or Epstein-Barr virus cell lines and NKG2A(+) natural killer cells, pre-treated with anti-human NKG2A, were rescued from disease progression. Human NKG2A(+) natural killer cells reconstituted in immunodeficient mice after transplantation of human CD34(+) cells. These natural killer cells are able to kill engrafted human primary leukemia or Epstein-Barr virus cell lines by lysis after intraperitoneal administration of anti-human NKG2A. Thus, this anti-NKG2A may exploit the anti-leukemic action of the wave of NKG2A(+) natural killer cells recovering after hematopoietic stem cell transplants or adoptive therapy with natural killer cell infusions from matched or mismatched family donors after chemotherapy for acute leukemia, without the need to search for a natural killer cell alloreactive donor.

Copyright© Ferrata Storti Foundation.

Figures

Figure 1.

HLA-E expression on acute leukemia cells. (A–C) HLA-E expression on AML cells from three patients. (D–F) HLA-E expression on ALL cells (1 T-ALL and 2 B-ALL) from three patients.

Figure 2.

In vitro treatment with anti-human NKG2A monoclonal antibody reconstitutes NKG2A+ NK cell lysis against HLA-E+ normal and neoplastic lymphohematopoietic cells. (A) Percentage lysis of KIR ligand-mismatched HLA-E+ B and T cells, monocytes, dendritic cells, EBV cell lines, CLL, T-ALL, AML and MM cells mediated by single KIR+ alloreactive NK clones at the E/T 15:1 in a standard 51Cr release cytotoxicity assay. (B) Percentage lysis of HLA-E+ B and T cells, monocytes, dendritic cells, EBV cell lines, CLL, T-ALL, AML and MM cells mediated by activated and cultured in IL2 NKG2A+/KIR− NK cells at the E:T of 15:1 after treatment with anti-human NKG2A monoclonal antibody (10 μg/1×106 cells/mL) in a standard 51Cr release cytotoxicity assay. Lysis mediated by NKG2A+ NK cells after treatment with anti-human NKG2A monoclonal antibody is comparable to lysis mediated by single KIR+ alloreactive NK cell clones. Each cytotoxicity assay was repeated with three targets for each category of cells and the mean ± SD is shown.

Figure 3.

Pre-treatment of human NKG2A+ NK cells with the anti-human NKG2A monoclonal antibody prevents engraftment of human EBV cell lines and AML cells and cures engrafted disease in NOD-SCID mice. (A) One million NKG2A+/KIR− NK cells were pre-treated with anti-human NKG2A monoclonal antibody (10 μg) and co-infused with EBV cell line (■) or AML (○) expressing HLA-E at an E:T of 1:12. Control mice were co-infused with isotype control antibody-pretreated NKG2A+/KIR−NK cells and EBV cell line (●) or AML (▲) expressing HLA-E at an E:T of 1:12. Mice co-infused with human EBV cell lines or human AML cells and treated with isotype control antibody-pretreated NKG2A+ NK cells died of disease progression. The anti-human NKG2A monoclonal antibody pre-treatment prevented disease engraftment and all mice survived. We pooled results of eight experiments with four mice per group for each experiment. (B) Mice engrafted with AML or EBV cell lines (20–30% of bone marrow infiltration) were infused with escalating doses of NKG2A+ KIR− NK cells, pre-treated with anti-human NKG2A monoclonal antibody (10 μg/1×106 NK cells). Control mice were co-infused with isotype control antibody-pretreated NKG2A+ KIR− NK cells and EBV cell line or AML cells. At least 3×106 NKG2A+ KIR− NK cells pre-treated with anti-human NKG2A monoclonal antibody cured 80% of mice with EBV or AML. Treatment of engrafted mice with at least 4×106 pretreated NKG2A+NK cells rescued 100% of mice affected by EBV (■) or AML (○). Mice engrafted with human EBV cell lines (●) or human AML cells (▲) and infused with more than 4×106 isotype control antibody-pretreated NKG2A+ NK cells died of disease progression. The anti-human NKG2A monoclonal antibody pre-treatment cured engrafted diseases. We pooled results of eight experiments with four mice per group for each experiment.

Figure 4.

In vivo treatment with the anti-human NKG2A monoclonal antibody rescues NSG mice engrafted with human CD34+ hematopoietic stem cells and HLA-E+ human AML cells or an EBV cell line. After 3.5 Gy total body irradiation, mice were infused with 10×106 human CD34+ hematopoietic stem cells. After 20 days they were infused with an EBV cell line or AML cells. When NKG2A+ NK cells differentiated from CD34+ cells, mice were treated with anti-human NKG2A monoclonal antibody. Mice that received 250 μg (○) or 300 μg (●) of anti-human NKG2A monoclonal antibody survived, control mice (isotype control antibody) (■) or mice that received 200 μg of the antibody (▲) succumbed to EBV lympho-proliferative disease (A) or AML (B). NKG2A+ NK cells ablated the EBV cell line in bone marrow* (C) and spleen (E) and AML cells in bone marrow* (D) and spleen (F). The normal human CD45+ hematopoietic population, which developed from CD34+ cells, was transiently depleted after administration of human anti-NKG2A antibody. We pooled results of three experiments with five mice per group for each experiment. * Bone marrow cell numbers are from two femurs per mouse.

Figure 5.

Treatment with the anti-human NKG2A monoclonal antibody transiently depleted HLA-E+ autologous myeloid, B, T, NK and DC subpopulations in NSG mice engrafted with human CD34+ hematopoietic stem cells. After 3.5 Gy total body irradiation, mice were infused with human CD34+ hematopoietic stem cells. One month after, when NKG2A+ NK cells differentiated from CD34+ cells reached a plateau value, mice were treated with 300 μg of anti-human NKG2A monoclonal antibody. Transient depletion of human myeloid, B, dendritic, and NK cell subpopulations in the bone marrow* (A) and spleen (B) and double negative (DN), single CD8+ or CD4+, CD4+/CD8+ double positive (DP) thymocytes (C) was followed by recovery of all cell subsets within 10 days. We pooled results of three experiments with five mice per group for each experiment. *Bone marrow cell numbers are from two femurs per mouse.