Ex vivo-expanded natural killer cells demonstrate robust proliferation in vivo in high-risk relapsed multiple myeloma patients

Abstract

Highly activated/expanded natural killer (NK) cells can be generated by stimulation with the human leukocyte antigen-deficient cell line K562, genetically modified to express 41BB-ligand and membrane-bound interleukin (IL)15. We tested the safety, persistence, and activity of expanded NK cells generated from myeloma patients (auto-NK) or haploidentical family donors (allo-NK) in heavily pretreated patients with high-risk relapsing myeloma. The preparative regimen comprised bortezomib only or bortezomib and immunosuppression with cyclophosphamide, dexamethasone, and fludarabine. NK cells were shipped overnight either cryopreserved or fresh. In 8 patients, up to 1×10⁸ NK cells/kg were infused on day 0 and followed by daily administrations of IL2. Significant in vivo expansion was observed only in the 5 patients receiving fresh products, peaking at or near day 7, with the highest NK-cell counts in 2 subjects who received cells produced in a high concentration of IL2 (500 U/mL). Seven days after infusion, donor NK cells comprised >90% of circulating leukocytes in fresh allo-NK cell recipients, and cytolytic activity against allogeneic myeloma targets was retained in vitro. Among the 7 evaluable patients, there were no serious adverse events that could be related to NK-cell infusion. One patient had a partial response and in another the tempo of disease progression decreased; neither patient required further therapy for 6 months. In the 5 remaining patients, disease progression was not affected by NK-cell infusion. In conclusion, infusion of large numbers of expanded NK cells was feasible and safe; infusing fresh cells was critical to their expansion in vivo.

Figure 1. Protocol schema

Bortezomib (Bor) was given (1.0mg/m², day −9, −6, −2) to enhance NK cell therapy by down-regulating expression of HLA Class I, and up regulating TRAIL receptor on the myeloma cell surface. ^,, Subjects 1, 2, 3, 5 and 8 also received immunosuppression with cyclophosphamide (Cy) 60 mg/kg IV on day −7, dexamethasone (Dex) 40 mg PO days −6 to −3, and fludarabine (Flu) 25 mg/m² IV days −6 to −2 to reduce T regs and to prevent early rejection of infused NK cells. One administration of 2×10⁷–1×10⁸/kg of NK cells was given on day 0 and followed by 13 doses of daily IL2 (3×10⁶ U, SC) to support the persistence and activity of NK cells in vivo.

Figure 2. Allo-NK cells proliferated further after infusion and circulating NK retained lytic activity against myeloma cells

A. The absolute number of NK cells per μL PB increased significantly post-infusion in subject 5. B. Chimerism studies confirmed that circulating PBMC in subject 5 were of donor origin. C. NK cells isolated from day 5 and day 7 post-infusion PB samples avidly killed subject 5 myeloma cells and K562 cells (⁵¹Cr release assay). D. Significantly higher NK cell levels were observed in the PB and BM of subject 8 who received NK produced in high IL2 (500 U/mL). E. The vast majority of post-infusion cells were of donor origin. F. Circulating NK cells obtained 14 days after infusion from the PB of subject 8 killed OPM2 myeloma and K562 cells (subject 8 myeloma was not available).

Figure 3. Auto-NK cells exhibited robust in vivo expansion, trafficked into the marrow, and exerted enhanced killing of auto-targets when HLA/KIR interactions were blocked or when ADCC was induced

A, C. Substantial in vivo expansion of NK cells was observed for subjects 4 and 6 who received fresh auto-NK cell products produced in 10 U/mL IL2. B, D. Circulating NK cells obtained from the PB 5 days after infusion did not induce significant in vitro killing of auto-myeloma cells although killing of K562 was high. E. The highest NK cell counts post infusion were observed in subject 7, who received NK produced in 500 U/mL IL2. F. Subject 7 myeloma cells were not available for in vitro assays until day 89; however OPM2 myeloma cells and K562 were avidly killed by BM-derived NK cells obtained 5 days after infusion. G. IHC staining further confirmed the presence of CD57⁺ lymphocytes in the BM of subject 7. Magnification is 400x. H. Enhanced NK cell mediated killing of auto-myeloma from subjects 4, 6, and 7 was achieved by inducing ADCC with elotuzumab (Elo) or blocking ligation of inhibitory receptors with antibodies to HLA Class I and KIR. E:T Ratio is 10:1.

Figure 3. Auto-NK cells exhibited robust in vivo expansion, trafficked into the marrow, and exerted enhanced killing of auto-targets when HLA/KIR interactions were blocked or when ADCC was induced

A, C. Substantial in vivo expansion of NK cells was observed for subjects 4 and 6 who received fresh auto-NK cell products produced in 10 U/mL IL2. B, D. Circulating NK cells obtained from the PB 5 days after infusion did not induce significant in vitro killing of auto-myeloma cells although killing of K562 was high. E. The highest NK cell counts post infusion were observed in subject 7, who received NK produced in 500 U/mL IL2. F. Subject 7 myeloma cells were not available for in vitro assays until day 89; however OPM2 myeloma cells and K562 were avidly killed by BM-derived NK cells obtained 5 days after infusion. G. IHC staining further confirmed the presence of CD57⁺ lymphocytes in the BM of subject 7. Magnification is 400x. H. Enhanced NK cell mediated killing of auto-myeloma from subjects 4, 6, and 7 was achieved by inducing ADCC with elotuzumab (Elo) or blocking ligation of inhibitory receptors with antibodies to HLA Class I and KIR. E:T Ratio is 10:1.

Figure 4. Immunophenotype of NK cells pre-expansion, post-expansion, and post-infusion (subject 7)

CD56 histogram overlays are gated on lymphocytes, all others are gated on CD3⁻CD56⁺ lymphocytes. Thin lines are isotype controls and thick lines are the indicated marker. The samples shown are the apheresis product analyzed the day of collection (Aph), the clinical product post-shipping (GMP NK), small scale research grade NK generated at UAMS (Res Grade NK) and PB samples obtained at the post-infusion time points indicated.

Figure 5. Serum levels of IL15, IL5, IL10, and TGFβ during the treatment course

A. Circulating levels of IL15 were elevated in subjects who received Flu (N=5) compared to subjects who did not receive immunosuppression (N=3). B–D. IL5, IL10, and TGFβ levels during the treatment course for subjects 4, 6, 7 and 8 as indicated. Markers are open for auto-NK cell recipients and closed for allo-NK cell recipients. Cytokine levels for subjects who received immunosuppression with fludarabine (Flu) are depicted with dotted lines. Subject 5 was omitted from the IL5, IL10, TGFβ analysis as IL2 was stopped on day 5.

Figure 6. Changes in T cell and T reg cell frequency

Markers are open for auto-NK cell recipients and closed for allo-NK cell recipients. A. Shown is the fold T cell change from the pre-chemotherapy value for the indicated subjects. B. The frequency of T reg cells was determined for subjects with sufficient circulating T cells (auto-NK cell recipients who did not receive immunosuppression). CD3⁺CD4⁺CD25⁺Foxp3⁺ T reg cells increased during the IL2 course, peaking at day 14. C. The NK:T reg ratio was higher than day 0 at days 5, 9, and 30 but not on day 14 when T reg levels had peaked and NK cell levels had begun to decrease. D. PBMC obtained from time points in which T reg cell levels were increased from baseline (post NK infusion days 9 or 14) were thawed and then whole PBMC (CD25 replete) and PBMC depleted of CD25⁺ cells using anti-CD25 antibody-coated magnetic beads were stimulated with irradiated K562-mb15-41BBL cells in the presence of IL2 (300 U/mL). NK cells were enumerated 10 days later by multiplying the viable cell count in the cultures by the NK cell percentage as determined by flow cytometry. Five replicate expansion cultures were set up for each condition. The mean ± the standard deviation is shown in addition to significance values; n.s., not significant.

Figure 7. K562-mb15-41BBL-stimulated NK cells are sensitive to TGFβ induced suppression

A. Stimulation of fresh whole PBMC obtained on protocol days 7, 9 and 14 (subjects 7 and 8) with irradiated K562-mb15-41BBL cells resulted in a 3.0 – 4.9 fold increase in NK cells after 5 days of culture. Lower NK cell expansion was observed in replicate cultures in which 5ng/mL recombinant human TGFβ was added. B. TGFβ cultures had significantly lower IFNγ production compared to vehicle cultures. C. Cell surface expression of NKG2D, DNAM1, NKp30, and NKp46 were also reduced after TGFβ treatment. Solid lines are vehicle, dashed lines are TGFβ, thin lines are isotype control. One representative example of 6 experiments is shown. D. Cytolytic activity was diminished in NK from TGFβ cultures. E. Phosphorylation of SMAD3 was observed in highly purified NK cells from the pre-NK infusion apheresis product (resting, RNK) and expanded NK cells treated with TGFβ, but not in expanded NK cells treated with vehicle (cells were obtained from subject 7). ENK, expanded NK.