Stimulation with K562 Cells Transfected with 4-1BBL and IL-15 Expands and Activates Natural Killer (NK) Cells with Specific Cytotoxicity for Multiple Myeloma (MM).

Introduction: Although stem cell transplant (PBSCT) supported high dose chemotherapy can induce remission in MM patients with high risk features (e.g. high CKS1-B expression and abnormal cytogenetics) there is a high rate of relapse. Immunologic therapies can augment high dose chemotherapy by eradicating chemo-resistant disease. We have previously established that KIR-ligand mismatched NK cells from haplo-identical donors are cytotoxic to primary MM cells. In this study we investigate whether co-incubation of NK cells with irradiated K562 cells transfected with 41BBL and membrane-bound IL15 (K562-41BBL-mIL15) could expand NK cells and augment cytotoxicity to primary MM cells.

Methods: Peripheral blood mononuclear cells (PBMC) of normal donors were co-cultured with irradiated K562-41BBL -mIL15 at a 1.5:1 ratio with varying concentrations of IL2 and re-stimulated on day 7. The cultures were then analyzed for proliferation, expansion, immunophenotype and cytotoxicity.

Results: By testing a range of IL2 concentrations (10–300 IU/ml) we first established that 100IU of IL2/ml induced optimal proliferation in H³ thymidine uptake assays and this concentration was used in all subsequent experiments. After two weeks PBMC co-cultured with K562-41BBL-mIL15 were enriched for NK cells (93%) and contained few T cells (0.8%) compared to PBMC cultured with IL2 alone (NK cells 11.9% and T cells 58.7% respectively). In absolute numbers there was as 60-fold (±0.6) expansion of NK cells in the K562-41BBL -IL15 cultures compared to only a 1.5-fold expansion of NK cells cultured with IL2 alone. NK cells were analyzed by flow cytometry for expression of KIR2DL1, KIR2DL2/3, and KIR3DL1 pre- and post-incubation with K562-41BBL-mIL15 and we observed no change in NK immunophenotype. ⁵¹Cr release assays of expanded NK cells showed specific killing of K562 cells (84±4%), the MM cell line U266 (88±3%), and primary MM cells (58±9%), without killing of non-myeloma patient cells (PHA-blasts) or autologous cells at a ratio of 7 NK effectors to 1 target cell. Expanded NK cells achieved much higher specific lysis and at lower E:T ratios of primary MM cells when compared side-by-side with non-expanded NK cells.

Conclusion: K562-41BBL-mIL15 transfectants stimulated vigorous expansion of NK cells without expanding T lymphocytes. Analysis of the KIR repertoire showed that there was no change in NK cell subpopulations after expansion. The expanded NK cells were highly active and killed patient MM cells better than non-expanded NK cells without significant lysis of normal cells. Expanded/activated NK cells may prove especially helpful in treating patients with relapsed/refractory MM who have a high tumor burden.