Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein

Abstract

Haploidentical natural killer (NK) cell infusions can induce remissions in some patients with acute myeloid leukemia (AML) but regulatory T-cell (Treg) suppression may reduce efficacy. We treated 57 refractory AML patients with lymphodepleting cyclophosphamide and fludarabine followed by NK cell infusion and interleukin (IL)-2 administration. In 42 patients, donor NK cell expansion was detected in 10%, whereas in 15 patients receiving host Treg depletion with the IL-2-diphtheria fusion protein (IL2DT), the rate was 27%, with a median absolute count of 1000 NK cells/μL blood. IL2DT was associated with improved complete remission rates at day 28 (53% vs 21%; P = .02) and disease-free survival at 6 months (33% vs 5%; P < .01). In the IL2DT cohort, NK cell expansion correlated with higher postchemotherapy serum IL-15 levels (P = .002), effective peripheral blood Treg depletion (<5%) at day 7 (P < .01), and decreased IL-35 levels at day 14 (P = .02). In vitro assays demonstrated that Tregs cocultured with NK cells inhibit their proliferation by competition for IL-2 but not for IL-15. Together with our clinical observations, this supports the need to optimize the in vivo cytokine milieu where adoptively transferred NK cells compete with other lymphocytes to improve clinical efficacy in patients with refractory AML. This study is registered at clinicaltrials.gov, identifiers: NCT00274846 and NCT01106950.

© 2014 by The American Society of Hematology.

Figures

Figure 1

Clinical trial schema. Patients received fludarabine 25 mg/m2/day intravenously (IV) daily (days −6 through −2) and cyclophosphamide 60 mg/kg/day IV (days −5 and −4) to lymphodeplete the recipient and facilitate homeostatic expansion of allogeneic NK cells. One (n = 11) or 2 doses (n = 4) of IL2DT, 12 (n = 11) or 18 mg/kg (n = 4) IV, were added at day −1 ± −2 to deplete Treg. NK cell products were administered by IV infusion on day 0 followed by subcutaneous IL-2 (9 × 106 units) starting 4 hours after NK cell infusion and given every other day for 6 doses to facilitate NK cell survival and expansion in vivo. Unseparated PB donor chimerism by STR and lymphocyte subsets were analyzed at days 7, 14, and 28. Bone marrow (BM) was analyzed for leukemia clearance at days 14 and 28 to assess disease status according to World Health Organization criteria. Toxicity and adverse events were classified according to National Cancer Institute Common Terminology Criteria for Adverse Events V 3.0. The primary prospective end point of the study was successful in vivo donor NK cell expansion defined as measurement of >100 donor NK cells/μL of PB at day +14 after NK cell infusion [(absolute lymphocyte count/μL) × (% of lymphocyte gate that are CD56+/CD3− NK cells) × (% donor chimerism using standard short tandem repeat testing)]. We evaluated BM at day 28 and used standard definitions of CR, CRp (<100 000 platelet count/μL), and CRi (<1000 absolute neutrophils/μL).

Figure 2

Treg depletion leads to NK-cell persistence and expansion that correlates with remission. (A) Successful in vivo donor NK cell expansion was observed in 80% of patients with Treg depletion (shown as percentage of PB lymphocytes; n = 6) compared with 0% in patients with high levels of Tregs (n = 9). (B) Rates of complete remission in patients with or without detectable donor NK cells in PB at day 7. (C) PB flow cytometry plot of selected subjects who demonstrated in vivo NK expansion at days 7 and 14.

Figure 3

In vivo expanded NK cells are potent killers, express high levels of NKG2A, and correlate with endogenous IL-15 prior to NK cell infusion. (A) Cytotoxicity of in vitro IL-2 activated NK cell products compared with PB NK cells isolated at day 14 after in vivo expansion. Cytotoxicity assay against K526 targets at various effector to target ratios. Cytotoxicity of the NK cell product (in gray; 15 infusion products; mean ± standard error of the mean [SEM]) prior to infusion compared with NK cells isolated from the PB at day 14 of those that expanded (black line; 4 subjects; showed mean ± SEM). (B) Expression of inhibitory receptors on NK cells in product and in vivo expanded NK cells at day 14. (C) Serum IL-15 levels at various time points after NK cell infusion. Comparison of IL2DT cohort patients with donor NK expansion (n = 4) vs no NK cell expansion (n = 11) and all patients not treated with IL2DT (n = 42, mean and standard deviation shown).

Figure 4

Cytokine-induced NK-cell proliferation is suppressed by allogeneic Treg cells. (A) Healthy donor purified NK cells were CSFE labeled and cultured alone or with UCB-derived Tregs at a ratio of 1:1. Suppression of proliferation of NK cells incubated with IL-2 (0.5 ng/mL), IL-15 (0.5 ng/mL), or a combination of IL-2 + IL-15 (0.25 ng/mL) was measured. Shown is 1 representative donor of 6 experiments. (B) Healthy donor NK cells and UCB-derived Tregs were coincubated with 0.5 ng/mL of IL-2 or IL-15 and cocultured at various Treg:NK cell ratios. Percent suppression of NK proliferation by Tregs was evaluated by CFSE dilution. Treg suppression of CD3 bead-stimulated PBMNC effector T-cell proliferation was measured as a control. Data are an aggregate of 5 separate experiments. NK cells and Tregs were coincubated with IL-2 or IL-15 for 4 days, and (C) IL-2 or (D) IL-15 in the supernatant was measured by enzyme-linked immunosorbent assay. Various cytokine concentrations (0.2, 0.5, and 10 ng/mL) and Treg:NK cell ratios (1:1, 2:1, 4:1, 8:1) were compared. Results from 5 NK cells donors are shown (mean and SEM). *P = .05 compared with NK cells alone.