Successful Haploidentical Hematopoietic Cell Engraftment Using a Non-Myeloablative Preparative Regimen Including Natural Killer (NK) Cells

Abstract

We have previously shown that adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in 27% of patients with refractory or relapsed acute myeloid leukemia (AML) [Miller et al., Blood 2005, 105 (8)]. Aiming to optimize NK cell expansion, which we hypothesize is required for therapeutic efficacy, we tested additional CD56-positive selection (N=10) versus the CD3-depletion method used for our earlier NK cell infusions (N=10). Donor-derived NK cells were not measurable immediately after infusion. Successful in vivo NK cell expansion, defined as >100 donor-derived NK cells/ml at 14 days (by VNTR chimerism and flow cytometry) was not improved with CD56-selection (11% vs. 11%; mean 131±3 NK cells/ml), and was associated with poorer outcomes (10% vs. 27% AML remissions). Because the remissions induced by adoptive NK cell transfer were not durable, we added a CD34+ stem cell infusion to create a nonmyeloablative haploidentical transplantation protocol for older and less fit patients. We also added radiation to the NK cell-based preparative regimen to further improve NK cell expansion. The lymphodepleting chemoradiation plus NK cell preparative regimen included fludarabine 25 mg/m2 × 5 (day -18 through day -14), cyclophosphamide 60 mg/kg × 2 (days -16 and -15), and 200 cGy of total body irradiation (twice a day on day -13). The NK cell product, prepared by cliniMACS (Miltenyi) CD3-depletion of a single leukapheresis collection from a haploidentical related donor, was incubated overnight in 1000 U/ml IL-2 and then infused on day -12 followed by 6 doses subcutaneous IL-2 (10 million units) given every other day to promote in vivo NK cell expansion. The mean NK cell dose was 1.85 × 107 cells/kg and the mean CD3+ cell dose was 4.8 × 104 cells/kg (maximum permitted 3 × 105 CD3+ cells/kg). A CD34-selected filgrastim-mobilized peripheral blood graft from the same donor (target dose >3 × 106 CD34 cells/kg) was given with Thymoglobulin 3 mg/kg days 0, +1 and +2 as the only additional immunosuppression. In the 13 patients treated to date a significantly higher rate of NK cell expansion (75% [9/12 evaluable]; mean 607±184 NK cells/ml) was achieved compared to the adoptive NK cell transfer regimen, which did not include radiation. Plasma IL-15, which is critical for NK expansion, was highest on day -12 (the NK infusion day) after the preparative regimen (64 ± 8 pg/ml [day -12] vs. 6 ± 1 pg/ml [baseline pre-chemo]; p <.0001). This adoptive NK cell plus allograft protocol led to 66% of relapsed or refractory AML patients (8/12 evaluable) clearing leukemia by day -1, with only one late relapse (day +93). Patients who did not clear leukemia (N=4) did not engraft, and it was not evaluable in 3 patients with early (pre-day +13) treatment related mortality (TRM). All others (N=6), engrafted quickly (defined by an absolute neutrophil count >500/ml and 100% donor chimerism: median 17 days [range 11–31]). None developed graft vs. host disease (GVHD), but infections were common (3 fatal EBV/PTLD; 1 Fusarium). To prevent EBV reactivation NK products are now CD19 depleted and patients receive prophylactic Rituxan to prevent PTLD. The other deaths were due to persistent disease (N=4) or relapse (N=1). One patient is alive in remission beyond day +100. No clear associations between killer immunoglobulinlike receptor (KIR) ligand mismatch between donor and recipient were detected. In this series of patients with refractory AML, addition of haploidentical NK cells to a nonmyeloablative haploidentical transplantation yields NK cell expansion in a majority of patients, achievement of complete remission, and quick engraftment without GVHD. This is a promising platform upon which to add other strategies aimed at improving disease free survival in patients with refractory AML.