NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC). NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials. The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days. Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15), which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL15. Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Until recently, the main limiting factor to the clinical application and efficacy of NK cells was the difficulty in obtaining sufficient cell numbers for adoptive transfer. Development of novel methods of expanding primary human NK cells ex vivo has renewed interest in NK cells for immunotherapy for cancer. [2][3][4][5][6] Expanded NK cells have enhanced expression of activating receptors, 4,7,8 that in turn improves their antitumor cytotoxicity. Where activating receptors did not sufficiently elicit an antitumor response, researchers augmented the antitumor effect of NK cells by expression of chimeric Ag receptors. 9-11 Ultimately, the success of NK cell adoptive immunotherapy for cancer depends not only on target recognition but also on homing of NK cells to the tumor target in vivo. Thus, the effector cells must express the appropriate chemokine receptors.Cancer cells express a wide array of chemokines and chemokine receptors that are instrumental in tumor survival 12 and metastatic spread. 13 Lymph nodes, particularly the tumor-draining nodes, are the foci of metastatic spread of tumors for a vast number of cancer types. 13,14 The expression of CCR7, a member of the G protein-coupled receptor family, on lymphocytes directs their homing to lymph node, coordinates primary immune responses, and induces peripheral immune tolerance. 15 CCR7 expression on tumor cells has been reported and shown to play a pivotal role in lymph node metastasis of various cancers such as breast, 16 pancreatic, 17 thyroid, 18 and colorectal 19 cancers; oral squamous cell carcinoma 20 ; melanoma 21 ; and lymphoma. 22 Lymph node involvement in these cancers is generally associated with poor prognosis.Peripheral NK cells express a variety of chemokine receptors such as CXCR1, CXCR3, and CXCR4 with subsets expressing CCR1, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR5, and CXCR6. Expression of CCR7 on NK cells can facilitate homing to lymph nodes, which, in the context of adoptive immunotherapy for various cancers, would offer a main advantage in targeting lymph node metastases. However, CD56 bright CD16 Ϫ NK cells, which primarily secrete cytokines, express CCR7, but CD56 dim CD16 ϩ NK cells, which are primarily responsible for cytotoxicity, do not. 23 In a previous study, we reported that expanded NK cells are predominantly of CD56 ϩ CD16 bright phenotype and did not express CCR7. 7 In this study, we sought to express CCR7 on expanded NK cells ex vivo to facilitate lymph node homing on adoptive transfer. Although investigators have used viral vectors to gene modify NK cell lines 10,24 and primary NK cells, 9,25 because of safety concerns over integrating viral vectors there has been a recent shift in emphasis toward nonviral methods of gene transfer, particularly nonintegrating, mRNA-based electroporation approaches. 11 However, electroporation of NK cells has been difficult in that the transfection efficiency and viability of NK cells are low, and high-throughput electroporation methods for gene modifying clinically relevant NK cell numbers are ...

Purpose Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAbs) has therapeutic potential for malignancies. We determined if large numbers of activated NK (aNK) cells can be grown ex vivo from peripheral blood mononuclear cells (PBMC) of children with high-risk neuroblastoma using artificial antigen-presenting cells (aAPC). Experimental Design Irradiated K562-derived Clone 9.mbIL21 aAPC were co-cultured with PBMC, and propagated NK cells were characterized with flow cytometry, cytotoxicity assays, Luminex® multi-cytokine assays, and a NOD/SCID mouse model of disseminated neuroblastoma. Results Co-culturing patient PBMC with aAPC for 14 days induced 2,363±443-fold expansion of CD56+CD3−CD14− NK cells with 83±4% purity (n=10). Results were similar with PBMC from normal donors (n=5). Expression of DNAM-1, NKG2D, FcγRIII/CD16 and CD56 increased 6±3, 10±2, 21±20, and 18±3-fold respectively on day 14 compared to day 0, demonstrating activation of NK cells. In vitro, aNK cells were highly cytotoxic against neuroblastoma cell lines, and killing was enhanced with GD2-specific monoclonal antibody ch14.18. When mediating cytotoxicity with ch14.18, release of TNFα, GM-CSF, IFNγ, sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC by aNK cells increased 4-, 5- 6-, 15-, 265-, 917- and 363-fold (151 to 9,121 pg/mL), respectively, compared to aNK cells alone. Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with thawed and immediately intravenously infused cryopreserved aNK cells compared to un-treated mice and was further improved when ch14.18 was added. Conclusion Propagation of large numbers of aNK cells that maintain potent anti-neuroblastoma activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18.