Infusion of haplo-identical killer immunoglobulin-like receptor ligand mismatched NK cells for relapsed myeloma in the setting of autologous stem cell transplantation

Abstract

Killer immunoglobulin-like receptor (KIR)-ligand mismatched natural killer (NK) cells play a key role in achieving durable remission after haplo-identical transplantation for acute myeloid leukaemia. We investigated the feasibility of transfusing haplo-identical, T-cell depleted, KIR-ligand mismatched NK cells, after conditioning therapy with melphalan and fludarabine, to patients with advanced multiple myeloma (MM) followed by delayed rescue with autologous stem cells. No graft-versus-host disease or failure of autologous stem cells to engraft was observed. There was significant variation in the number of allo-reactive NK cells transfused. However, all NK products containing allo-reactive NK cells killed the NK cell target K562, the MM cell line U266, and recipient MM cells when available. Post NK cell infusion there was a rise in endogenous interleukin-15 accompanied by increasing donor chimaerism. Donor chimaerism was eventually lost, which correlated with the emergence of potent host anti-donor responses indicating that the immunosuppressive properties of the conditioning regimen require further optimization. Further, blocking of inhibitory KIR-ligands with anti-human leucocyte antigen antibody substantially enhanced killing of MM cells thus highlighting the potential for modulating NK/MM cell interaction. Encouragingly, 50% of patients achieved (near) complete remission. These data set the stage for future studies of KIR-ligand mismatched NK cell therapy in the autologous setting.

Fig 1

Protocol schema ‘haplo-identical NK cell therapy combined with delayed autografting’. Conditioning therapy comprised fludarabine (Flu, 25 mg/m² on day −5 to day −2), dexamethasone (Dex, 40 mg/d on days −5 to −2), and melphalan (MEL, 140 mg/m² IV on day −1). Flu and Dex were given to deplete patient lymphocytes in order to prevent rejection of allogeneic NK cells. MEL 140 mg/m² was used for tumour reduction. NK cells from a haplo-identical KIR-ligand mismatched family donor were transfused on days 0 and +2. IL-2 was given daily to support the survival and expansion of NK cells in vivo. Autologous PBSCT was delayed to provide a window for donor NK cell product to kill residual myeloma cells.

Fig 2

All donor NK products killed KIR-ligand mismatched MM cells. Donor NK cells lysed MM cell targets lacking inhibitory KIR-ligands including patient MM cells (when available), with the exception of donor 7, who did not have allo-reactive NK cells. K562, patient MM cells, U266 MM cell line (homozygous C group 1 and HLA-Bw4 negative), and patient PHA blasts were employed as targets in a standard 4-h ⁵¹Cr release assay, at E/T ratios of <10:1. Patient primary MM cells were available for patients 2, 3, 6, 7 and 8. Specific lysis percentage was calculated as (test release − spontaneous release)/(maximal release − spontaneous release) × 100. All experiments were performed in triplicate wells and the mean ± SD were presented. One of three independent experiments was shown. All experiments were performed with the final NK cell product, which had been incubated overnight (products 1–4) or during cell processing with 300 IU/ml of IL-2 (products 5–10).

Fig 3

Blocking of HLA class I molecules increases NK cell-mediated lysis of primary MM cells. Anti-human HLA-ABC monoclonal antibody, clone W6/32, (10 μg/ml) was used in the blocking experiments. Control and blocked cells were employed as targets in the cytotoxicity assay, at E/T ratios of <10:1. Specific lysis percentage was expressed as (test release − spontaneous release)/(maximal release − spontaneous release) × 100. All experiments were performed in triplicate wells and the mean ± SD was present. One of three independent experiments was shown. Effectors comprised the corresponding haplo-identical donor’s final NK cell product after IL-2 incubation. Pt, patient.

Fig 4

Persistence of donor cells in vivo in patients post NK cell infusion. Persistence of donor cells was assessed by real-time PCR-based chimaerism assay analysing polymorphisms of HLA-DRB1 or short tandem repeat. The total WBC and lymphocyte count (×10⁹/dl) are indicated above each data point. Pt, patient; NA, not available.

Fig 5

Patient T cells responded to donor cells post NK cell infusion. Mean ± SD of stimulation index (SI) is shown. The data shown are representative of an average of four recipient-donor MLRs analysed. The composition of the responder cells at each day is shown for each time point to demonstrate that the increase in SI reflected an increase in actual T-cell responsiveness over time rather than a change in CD3⁺ T-cell composition. SI = CPM in cultures with donor NK cell product/CPM in cultures with autologous PHA blasts.

Fig 6

NK cell growth factor IL-15 was detected in vivo post NK cell infusion. Serum levels of IL-15 were measured by ELISA. Black squares represent the mean values ± SD of nine recipient-donor pairs studied. Open triangles represent the mean values ± SD of two patients treated with autologous PBSCT after standard melphalan 200 mg/m² conditioning therapy.