Novel treatment strategy with autologous activated and expanded natural killer cells plus anti-myeloma drugs for multiple myeloma

Abstract

This proof-of-concept single-arm open-label phase I clinical trial (NCT02481934) studied the safety and efficacy of multiple infusions of activated and expanded natural killer (NKAE) cells in combination with anti-myeloma drugs in multiple myeloma patients. It included five patients with relapsed or refractory MM who had received two to seven prior lines of therapy; NK cells were expanded for 3 weeks with K562-mb15-41BBL cells. Patients received four cycles of pharmacological treatment with two infusions of 7.5 × 10⁶ NKAEs/kg per cycle. NKAE generation, expansion, and NK monitoring was assessed using flow cytometry. Eighteen clinical-grade NKAE cell GMP-grade products were generated to obtain 627 × 10⁶ NKAEs (range: 315-919 × 10⁶) for the first infusion and 943 × 10⁶ (range: 471-1481 × 10⁶) for the second infusion with 90% (±7%) purity. Neutropenia grade II occurred in two patients and was related to chemotherapy. Of the five patients, four showed disease stabilization before the end of NKAE treatment, and two showed a 50% reduction in bone marrow infiltration and a long-term (>1 y) response. The NKAE cells had a highly cytotoxic phenotype and high cytotoxicity in vitro. Infused NKAE cells were detected in bone marrow and peripheral blood after infusions. Ex vivo expansion of autologous NK cells is feasible, NKAE cells are clinically active and the multiple infusions are well tolerated in patients with relapsed or refractory myeloma.

Keywords: Activated and expanded NK cells; cell therapy; clinical trial; immunotherapy; multiple myeloma.

Figure 1.

Characteristics of the activated and expanded natural killer cells (NKAEs). The characteristics of NKAEs expanded from the multiple myeloma patients in the NCT02481934 clinical trial were monitored every week by flow cytometry, and cell counts were also performed weekly. The results are reported as the mean value ± standard deviation of four independent expansion procedures from each patient. (A) The NKAE cell counts and (B) The reduction in T cell counts in the NKAE end products for the 18 expansion procedures. NKAE cell purity and T cell contamination percentages from each week are shown above its corresponding chart (as mean value ± standard deviation). (C) Representative dot plots of flow cytometry analyses of NKAEs during the expansion process. Each dot plot corresponds to each week of expansion (i: day 1, ii: day, 7, iii: day 14, and iv: day 21). (D) NKAEs overexpressed activating receptors and apoptosis ligands relative to NK cells before expansion. The data are reported as the median with interquartile range (IQR). (E) NKAEs had significantly better cytotoxic activity than NK cells against U-266 myeloma cells. The data are reported as the mean value ± standard deviation (n > 3). The p-values are from t-tests comparing the percentage of U-266 cells lysed by the NK cells pre-expansion with those lysed by NKAEs (NK cells post-expansion).

Figure 2.

Assessment of the response of multiple myeloma patients to the infusion of activated and expanded natural killer cell (NKAEs). (A) The response to NKAE infusion was determined by monitoring the peripheral blood monoclonal protein levels (g/dL) until relapse and the bone marrow plasma cell infiltration (BMI) before and after NKAE cell therapy. The scale on the x-axis is months. Blue arrows indicate NKAE cell infusions, and gray arrows indicate when bone marrow aspirations were performed to determine BMI percentages. (B) The serum monoclonal protein levels and serum lambda chain levels (mg/mL) in patient 5 were compared before treatment and at the time of the best response.

Figure 3.

Monitoring activated and expanded natural killer (NKAE). NKAE cells were detected in peripheral blood samples by flow cytometry. Peripheral blood was obtained from the patients each cycle before each infusion, then after infusion, and then 1 h, 1 d, and 3 d after infusion, as possible. (A) The expression of NK cell surface receptors before and after treatment with NKAE cells. The results are reported as the median with interquartile range for five independent procedures. (B) Representative histograms showing the expression of NK cell receptors from a single patient before and after NKAE cell treatment. (C) NK cells were detected on peripheral blood Wright stained smears. (D) Cytokine serum levels were analyzed before treatment and on days 1, 3, 7, 15, and 28 of NKAE cell treatment. The IL-10 and IFNγ levels in five patients are shown as the fold changes compared to the screening (pre) cytokine concentrations. (E) Representative flow cytometry data of lymphocyte populations from a single patient during the first cycle. (F) The percentage of peripheral blood Treg CD4⁺CD25⁺CD127⁻ cells during treatment with NKAEs.

Figure 4.

Treatment procedures and timeline. Two weeks before each cycle of chemotherapy, 200 mL of peripheral blood was extracted from each of the five multiple myeloma patients to produce activated and expanded natural killer cells (NKAEs). The NKAEs were infused in the second and third weeks of expansion on days 1 and 8 of each of the four cycles of lenalidomide- or bortezomib-based treatment. The scale in the x-axis is days.