Expansion of highly cytotoxic human natural killer cells for cancer cell therapy

Abstract

Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to maximize the potential of this approach. We determined the capacity of the leukemia cell line K562 modified to express a membrane-bound form of interleukin (IL)-15 and 41BB ligand (K562-mb15-41BBL) to generate human NK cells with enhanced cytotoxicity. Seven-day coculture with irradiated K562-mb15-41BBL induced a median 21.6-fold expansion of CD56(+)CD3(-) NK cells from peripheral blood (range, 5.1- to 86.6-fold; n = 50), which was considerably superior to that produced by stimulation with IL-2, IL-12, IL-15, and/or IL-21 and caused no proliferation of CD3(+) lymphocytes. Similar expansions could also be obtained from the peripheral blood of patients with acute leukemia undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells and their unstimulated or IL-2-stimulated counterparts showed marked differences. The expanded NK cells were significantly more potent than unstimulated or IL-2-stimulated NK cells against acute myeloid leukemia cells in vitro. They could be detected for >1 month when injected into immunodeficient mice and could eradicate leukemia in murine models of acute myeloid leukemia. We therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions, generating large numbers of highly cytotoxic NK cells. The results that we report here provide rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy of cancer.

Figures

Figure 1. Activation and expansion of NK cells from peripheral blood

(A) Schema of the NKAES method. Irradiated K562-mb15-41BBL cells are mixed with peripheral blood mononuclear cells (PBMC) at a 1 : 1.5 ratio. (B) NKAES-NK cell expansion from mononuclear cells from 50 healthy donors. Fold recovery of CD56+CD3- cells after 7 days of culture relative to the number of input cells is shown; bar: median. (C) Ki67 and CD25 expression were examined after 5 days of NKAES culture, a stage at which cultures typically still contain a majority of non-NK cells as well as NK cells in their initial phase of expansion; >90% CD56+ cells expressed both markers, whereas CD56- cells remained mostly Ki67- and CD25-.

Figure 2. Gene expression analysis of unstimulated-, IL-2 stimulated- and NKAES-NK cells

Unstimulated-NK cells (n = 5) were studied immediately after immunomagnetic separation of CD56+CD3- cells. IL-2-stimulated- (n = 6) and NKAES-NK cells (n = 5) were studied after 7 days of culture. Two concentrations of IL-2 were used: 200 IU/mL (n = 3), and 6000 IU/mL (n = 3). (A) Unsupervised hierarchical clustering analysis resulted in 3 major clusters, each containing all unstimulated-, IL-2-stimulated- and NKAES-NK samples. IL-2- stimulated NK samples were in one major cluster regardless of the IL-2 concentration used (brown line, 200 IU/mL; green line, 6000 IU/mL). (B) Supervised clustering analysis of NKAES-NK cells compared with unstimulated NK cells. (C) Supervised clustering analysis of NKAES-NK cells compared with IL-2 stimulated cells. The top 1000 differentially expressed probe sets are shown (FDR < 0.02) in B and C.

Figure 3. Cell marker expression of unstimulated NK cells, NK cells stimulated with 6000 IU/mL IL-2 for 7 days and NKAES-NK cells

(A) Flow cytometry analysis of molecules differentially expressed by gene array analysis. Overlay histograms show results of 1 representative experiment out of 3. (B) Expression of natural cytotoxicity receptors (NKp30, NKp44, NKp46), NKG2D and its ligands (MICA/B, UBP1, ULBP2, and ULBP3).

Figure 4. Antileukemic activity of NKAES-NK cells against AML cells

(A) 4-hour cytotoxicity against leukemic cell lines with NK cells from 12 donors. Mean of four measurements is shown at each E : T ratio. (B) 4-hour cytotoxicity of NKAES-NK cells from 4 donors against AML cells from 2 patients. (C) Cytotoxicity against AML cells from 5 patients after 5 days of culture on mesenchymal cells. The mean ± SD cell killing at the indicated E : T ratios in triplicate cultures is shown.

Figure 5. Antileukemic activity of NKAES-NK cells compared with that of unstimulated and IL-2-stimulated NK cells

(A) Cytotoxicity of unstimulated and NKAES-NK cells (from the same donors; n = 12) was tested against leukemic cell lines. Mean results of two measurements after 4 hours of culture are shown. Bars: median. Similar comparisons performed between NKAES-NK cells and NK cells stimulated with 1000 IU IL-2 for 16 hours (from 3 donors) (B), or (C) 6000 IU/mL IL-2 for 7 days (2 donors). NK cells from the same donors were tested in each comparison.

Figure 6. Antileukemic activity of NKAES-NK cells in vivo

NOD/scid-IL2RGnull mice (n = 6) were injected with K562 cells expressing luciferase (2×105) i.p. Then, NKAES-NK cells (1×107) from the same donor were injected every 2 days i.p in 3 mice, from day 1 to day 11 after K562 injection. All 6 mice received IL-2 25000 IU i.p. daily for 3 weeks. (A) Leukemia cell growth was visualized through luciferin injection and Xenogen imaging (ventral is shown). Leukemia progressed in all 3 mice not treated with NK cells (top panels) and euthanized between day 37 and 44. By contrast, leukemia progression was not apparent in 2 of the 3 mice receiving NK cells which remain alive and leukemia-free 8 months after the beginning of the experiment; in a third mouse, leukemia became detectable on day 43 (the mouse was euthanized on day 70; bottom panels). (B) Signal intensities (photons/second) detected in control (left) and NK-treated mice (right).