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

Veronika Bachanova, Sarah Cooley, Todd E. Defor, Michael R. Verneris, Bin Zhang, David H. McKenna, Julie Curtsinger, Angela Panoskaltsis-Mortari, Dixie Lewis, Keli Hippen, Philip McGlave, Daniel J. Weisdorf, Bruce R. Blazar and Jeffrey S. Miller

Data supplements

Article Figures & Data

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.

Tables

  • Table 1

    Patients, treatment, and product characteristics

    VariableCohort 1Cohort 2Cohort 3
    No. of patients enrolled321015
    Time period2003-200720052010-2011
    Patient age in years (range)46 (7-68 y)37 (5-65 y)51 (3-71 y)
    Patient gender (male)19 (59%)5 (50%)8 (55%)
    Marrow blasts (mean %)45% (range 2-98)36% (range 7-92)34% (range 8-69)
    Number of prior therapies (mean)433
    Prior HCT2 auto/4 allo00
    Recipient CMV+ status14 (44%)6 (60%)7 (45%)
    ConditioningCy/FluCy/FluCy/Flu
    No. of IL-2 doses (mean)4 (range 3-6)4 (range 3-6)4 (range 1-6)
    IL2DT receivedNoNoYes*
    KIR mismatch in GVHD direction6 (17%)5 (50%)8 (53%)
    Product processing methodCD3-CD3-CD56+CD3-CD19-
    Final product characteristics
    Dose of NC/kg2.5 ± 0.8 x1070.44 ± 0.09 × 1074.7 ± 1.8 × 107
    Dose of NK cells/kg infused0.96 ± 0.3 × 1070.34 ± 0.05 x1072.6 ± 1.5 × 107
    Percentage NK cells39 ± 9%75 ± 6%54 ± 16%
    Dose of T-cells/kg14 × 1046.2 × 1049.7 × 104
    Percentage T cells0.7%1.3%0.3%
    • CD3, CD3 depletion; CD3CD56+, CD3 depletion followed by CD56-positive selection; CD19, CD19 negative selection; CMV, cytomegalovirus; NC, nucleated cell dose.

    • * Doses of 12 to 18 μg/kg × 1 (n = 11) or 2 doses (n = 4) were used.

  • Table 2

    Treatment details, correlative analysis, and patient outcome

    No.Age (years)Prior diseaseWBC*Cytogenetics/FISHMolecularMarrow blasts*No. of prior therapiesDisease statusKIR ligand mismatchPB donor chimerism at day 7Donor PB NK cells at day 14 (cells/μL)PB percentage of Tregs at day 14Disease response at day 28Time to death/time of survival (mo)Survival status
    166MDS82t(1;3),del 5q-EGFR96%2PIFNo95%123900.02%CRi1.1Dead
    259MDS0.2Complex, del 7p-EGFR34%3PIFNo95%4840.1%PD0.9Dead
    349RAEB-255NormalFlt3 ITD49%3PIFNo13%071%CR10Dead
    451ET/MDS10del 5q-, del 20q-EGR140%3PIFNoQNS049%PD0.8Dead
    55Primary AML110t(9;11)MLL95%5PIFYes0%027%PD1.1Dead
    651Primary AML32Monosomy 7EVI181%4PIFYes34%241.1%PD2.5Dead
    771MDS0.2Complex, del20q-MYBL26%3RelapseYes68%017%CR9.4Alive
    864MPD53Monosomy 7EGFR9%5PIFNo92%5320.06%CRi2.6Dead
    957MPD0.7NormalJak2 +, Flt3 ITD24%2PIFYesQNSNo11%CRi12Alive
    1037Primary AML2.2NormalFlt3 ITD10%3PIFNo33%No28%CRp12Alive
    1113MDS225Trisomy 6Flt3 ITD7%3PIFNo42%No10%CR10.2Dead
    1254Primary AML5.6t(12;14)ETV636%4RelapseYes0%NoNAPD0.4Dead
    135Primary AMLNAt(9;11)MLL33%4RelapseYes0%No16%PD1.1Dead
    1416Primary AML50t(3;22) del 9qn/a85%6PIFYes99%14700%PD1.2Dead
    1573Primary AML2.5Trisomy 1PBX124%2RelapseYes70%No20%CRp12Alive
    • ET, essential thrombocythemia; MPD, myeloproliferative disorder; NA, not available; PD, progressive disease; PIF, primary induction failure; QNS, quantity not sufficient; RAEB, refractory anemia with excess blasts; WBC, white blood cell.

    • * At time of therapy (in 106 cells/μL).

    • Includes prior demethylation agent.

    • Subsequent allogeneic donor stem cell transplant.