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, 97 (9), 1348-56

Highly Activated and Expanded Natural Killer Cells for Multiple Myeloma Immunotherapy

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Highly Activated and Expanded Natural Killer Cells for Multiple Myeloma Immunotherapy

Tarun K Garg et al. Haematologica.

Abstract

Background: Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma.

Design and methods: OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction.

Results: Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold, respectively, without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D, natural cytotoxicity receptors, and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred, expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion, persisted up to 4 weeks, were readily detectable in the human bone, inhibited myeloma growth and protected bone from myeloma-induced osteolysis.

Conclusions: These studies provide the rationale for testing expanded natural killer cells in humans.

Figures

Figure 1.
Figure 1.
Exp-NK cells have an activated phenotype. Flow cytometry confirms the increased cell surface density of NKG2D, NKp30, NKp44, CD26, CD56, CD54, CD69, CD70, and the chemokine receptor CXCR3. Open peaks represent non-exp-NK, shaded peaks represent exp-NK. One representative result from 12 experiments is shown.
Figure 2.
Figure 2.
Exp-NK cells (unselected, purity >92%) kill MM cells and this killing is mediated by the perforin pathway and critical activating receptor-ligand interactions. (A) Exp-NK cells kill K562 cells and the MM cell line U266 better than non-exp NK cells (P<0.001; the mean and SEM for nine assays are shown). (B) Exp-NK cells derived from HD kill primary MM cells while killing of patients’ phytohemagglutinin blasts remains low (P<0.001; the mean and SEM for six assays are shown). (C) Exp-NK cells derived from MM patients can induce significant killing of autologous MM cells (P<0.001; the mean and SEM for eight assays are shown). (D) Killing of primary MM cells by exp-NK is similar whether NK cells are derived from MM patients or HD (P>0.2), whereas killing is lower when targets are autologous. (E) HD-derived exp-NK cells kill primary MM cells via a perforin pathway. Blocking effectors with anti-TRAIL or anti-FAS-L antibodies did not significantly reduce the level of killing whereas the addition of the perforin inhibitor concanamycin A (CMA) to the assay reduced killing appreciably. One of three representative assays is shown. Blocking critical NK cell receptors (F) or their ligands on MM cells (G) can inhibit killing of primary MM cells. The mean ±SEM for three independent experiments is shown for (F) and (G).
Figure 3.
Figure 3.
Human exp-NK cells significantly inhibit luciferase-transfected OPM2 myeloma tumor growth in the NOD/scid/IL2Rγnull-hu model. (A) Imaging of OPM2 tumors expressing luciferase illustrates the myeloma burden in mice receiving PBS (control) or a total dose of 160×106 exp-NK cells (given by four intravenous injections 48 h apart). Day 0 = date of NK cell injection #1. IL2 (100 U) was given intraperitoneally twice weekly to support NK cell survival in vivo. Three representative mice from each group are shown. (B) The fold tumor volume relative to baseline values was significantly lower in the cohort of mice that received 160×106 exp-NK cells than in either the control group or the group that received a lower NK cell dose (40×106). Each symbol represents one mouse in the different groups at the days indicated and median values are noted for each data set (−).
Figure 4.
Figure 4.
Exp-NK cell treatment protects bone from myeloma-induced osteolysis. On day 29, at the termination of the experiment depicted in Figure 3, implanted fetal bones were excised and subjected to immunohistochemistry and micro-computed tomography (CT). H&E, TRAP, and osteocalcin staining revealed gross loss of bone, increased numbers of osteoclasts, and reduced numbers of osteoblasts in the control group (A–C) compared to those in the mice treated with 160×106 exp-NK cells (F–H). Micro-CT further confirmed the dramatic difference in implanted human bone structure between control and exp-NK cell–treated groups. Three-dimensional reconstructions of the bones are shown (D, I), as well as longitudinal sections through the midpoint from each specimen (E, J). Heavy bone resorption is clearly seen in the control group (D, E), while bone resorption is not prominent in the exp-NK cell–treated group (I, J).
Figure 5.
Figure 5.
Exp-NK cells inhibit primary MM cell growth in a NOD/scid/IL2Rγnull-hu model. ELISA for huIg demonstrates significant tumor growth inhibition in vivo at day 21. Each symbol represents one mouse and the median values are also shown (−).
Figure 6.
Figure 6.
Human exp-NK cells persist for up to 28 days, proliferate further in the peripheral blood of OPM2 myeloma-bearing NOD/scid/IL2Rγnull mice and can be detected in the MM tumor bed. (A) NOD/scid/IL2Rγnull-hu hosts bearing OPM2 tumors were dosed with 160×106 exp-NK (given by four i.v. injections 48 h apart) followed by either “low IL2” (100 U IL2 twice weekly, open bars), or “high IL2” (1000 U IL2 daily, filled bars). Peripheral blood was collected weekly and subjected to flow cytometry. Exp-NK cells were detectable 28 days after infusion in mice given high IL2. One representative set of dot plots is shown for days 7, 14, 21, 28 for a mouse receiving high IL2. *Exp-NK high IL2 versus low IL2 P<0.005. Day 0 = date of NK cell injection #1. (B) Exp-NK cells proliferated in vivo in NOD/scid/IL2Rγnull mice receiving high IL2. CFSE-labeled exp-NK cells (4×107) were injected i.v., and peripheral blood was obtained for flow cytometry analysis 6 days after injection. (C) Exp-NK cells were detectable in appreciable numbers in the MM tumor bed 28 days after adoptive transfer. In paraffin sections, exp-NK cells were stained brown (arrows) with anti-human CD57 and MM cells were stained red (arrowhead) with CD138 antibodies in conjunction with DAB and Fast red for dual immunohistochemistry staining. Nuclei are stained blue with hematoxylin.

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