Preparation of the NK cell product
The apheresis product was T cell- (CD3-) depleted using the Miltenyi Biotec CliniMACS® Cell Selection System and CD3 MicroBeads and reagent and cultured in serum-free media (X-VIVO 15) supplemented with 10% human AB serum and 1,000 U/mL IL-2. Following 8–16 hours incubation, cells were washed twice with a 5% human serum albumin. An aliquot of the cell product was analyzed by flow cytometry to determine the number of T, B and NK cells (using FITC, APC, PE and perCP conjugated antibodies against CD3, CD56, CD19, KIR, NKG2A, NKG2D, CD16, CD69 and tested in a 4-h Cr-release cytotoxicity assay against K562 cell line pre and post IL-2 incubation.
Patients received Xudarabine 25 mg/m2/day intravenously (IV) daily (days -6 to -2) and cyclophosphamide 60 mg/kg/days IV day -6 to induce immunosupression and lymphodepletion required to facilitate homeostatic expansion of allogeneic donor NK cells. Rituximab 375 mg/m2 IV was administered on day -8 and then weekly for 3 more doses (days -1, +6, +15) to enhance ADCC. Cell products were administered by intravenous infusion on day 0 followed by subcutaneous IL-2 10×106 units starting 4 hours after NK cell infusion and given every other day x6 doses to facilitate NK cell survival and expansion in vivo.
The high-dose chemotherapy and NK cell infusion did not induce unexpected toxicity. 5 of 6 patients completed all prescribed IL-2 doses. No patient developed prolonged marrow aplasia or GVHD. Non-hematologic toxicities were NCI CTCAE grade 1–3 rigors and fevers with the NK cell infusion (4 patients), skin redness/swelling at IL-2 injection site (4 patients), fatigue (3 patients), sepsis (1 patient), hypertension (1 patient), bigeminy related to electrolyte disturbance (1 patient) and tumor-induced airway obstruction (1 patient). In 1 patient, IL-2 was stopped after the fourth dose due to dyspnea with pulmonary infiltrates. The patients’ condition rapidly improved with antifungal therpy although no fungal infection was documented. We observed grade 3 neutropenia and thrombocytopenia in all patients.
Four patients achieved objective remissions;2 complete remissions (marginal zone lymphoma and follicular lymphoma), 2 partial remissions (transformed lymphoma and diffuse large B cell lymphoma) on day 28 which were maintained at the 2months post-therapy evaluation.
Two patients (marginal and follicular) with complete resolution of lymphoma on PET/CT also cleared their bone marrow of lymphoma at 2 months. Three responding patients underwent allogeneic donor HCT at 116, 70 and 62 days post-NK cell therapy with no evidence of disease progression at the time of transplant. The PR in the fourth responding patient (DLBCL) lasted for 123 days.
All patients demonstrated substantial increases in host-regulatory T cells (Treg) after NK cell and IL-2 therapy (180 ± 80 cells/μl vs. baseline: 58 ± 24 cells/ μl, p = 0.04) which may have limited donor cell expansion in vivo.
These findings suggest safety and feasibility of allogeneic NK cell therapy in patients with lymphoma; however host Treg and inadequate immunodepletion may contribute to a hostile milieu for NK cell survival and expansion.
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Scientific article publishing date :8/3/2010
Immucura identifier BSC21_222EN