Adoptive transfer of NK cells engineered to express new or augmented functions represent an interesting avenue for the treatment of various high-risk malignancies in which conventional options have failed (Figure 1). Examples include B-chronic lymphocytic leukemia (B-CLL) (3), multiple myeloma (4), and also tumors of non-hematopoietic origin such as breast cancer (5), melanoma, or renal cell carcinoma (6).

Early studies reporting the adoptive transfer of CD56⁺ bead-selected autologous NK cells into patients with metastatic cancers, in combination with high doses of IL-2 demonstrated feasibility yet poor clinical efficacy (7). Patients experienced severe toxic side effects due to the high doses of IL-2. In addition to activation-induced NK cell death, NK cell function may have been inhibited due to regulatory T cell expansion in response to high IL-2 doses (8, 9). Reducing the daily IL-2 doses after NK cell transfer resulted in limited clinical success (10). Several strategies are under investigation to overcome this hurdle [reviewed in Ref. (11)]. Another mechanism by which autologous NK cells are inhibited is by self-HLA molecules. Therefore “releasing the brakes” with anti-KIR antibodies such as Lirilumab^® that targets the inhibitory KIR receptors on NK cells could be one approach (12–14).

A number of arguments support an important role for donor-derived NK cells in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Following the administration of either myelo-ablative (15) or reduced-intensity (15–17) conditioning regimen and allo–HSCT, the rapid reconstitution of high numbers of circulating and phenotypically defined NK cells is associated with better clinical outcome. The recovery of various functions for donor-derived cells may be further modulated and improved in vivo with additional intervention (18).

Transplantation of high doses of immune-selected CD34⁺ cells collected from haploidentical donors after myelo-ablative conditioning regimen has provided a setting which demonstrates that “KIR-incompatibility” was associated with lower incidence of disease relapses, at least for AML (19). Transplantation of T-replete marrow or blood cell grafts obtained from haploidentical donors, using modified immune-suppressive conditioning regimen such as those including posttransplant cyclophosphamide, represent a more widely applicable procedure, in which to further explore the potential contribution of alloreactive NK cells in posttransplant clinical events. Unexpectedly, a recently published report suggests that, in this context, the presence of recipient class I ligands to donor KIR receptors confers some protection to the recipient against leukemia relapse, an observation that needs further confirmation and would imply a role for killer activating receptors (KAR) as much as for KIR (20). The role of alloreactive NK cells remains more elusive in the context of HSCT performed from other categories of donors. Expression of specific KIR receptors in HLA-matched unrelated donors was demonstrated to produce superior or inferior clinical outcomes in recipients, depending on donor–recipient combinations (21–23).

Adoptive transfer of allogeneic NK cells either with a stem cell graft ex vivo depleted of immune effectors or as a substitute to posttransplant “donor lymphocyte infusions” (DLIs) is thus appealing as a way to improve engraftment, immune reconstitution, and antitumor activity with reduced chances of triggering graft-versus-host disease (GVHD) (24). Results of a small number of clinical trials have been reported so far, demonstrating the feasibility of manufacturing allogeneic NK cells from matched related, matched unrelated, or mostly from haploidentical donors (25–29). Although allogeneic NK cell infusions were generally reported as safe, a recent publication describes the clinical outcome of a small cohort of pediatric patients treated for non-hematological high-risk malignancies and a high proportion of aGVHD triggered by HLA-matched donor-derived NK cells (30). Mostly, these limited clinical results suggest that additional improvements are needed either during the ex-vivo manufacturing process (31) or after infusion of manufactured NK cells (25) to improve long-term persistence and activity in vivo.

In the setting of allogeneic NK-DLI, donor selection can affect the clinical outcome of NK cell therapy, since certain KIR, HLA, and FcγR polymorphisms influence NK cell function (32, 33). KIR typing can be genotypic, classifying donors on the basis of gene expression of activating and inhibitory KIR (34), thereby assigning them scores to select “preferable donors” (35–37). Additionally, KIR typing can be phenotypic, assessing surface protein expression of KIRs (38), adding another layer of complexity to the selection of “preferable donors.” KIR “allele-typing” is a recent addition to the donor selection algorithm, whereby alleles that possess better functional properties (stronger licensing capability and more durable surface expression upon ligand binding) are favored (22, 39). Typing FcγR polymorphism is relevant in NK cell therapy settings that use monoclonal antibodies to enhance NK cell activation and consequently empower their antibody-dependent cytotoxicity (ADCC) properties (40, 41). All these strategies have helped define “preferable donor” profiles.

Natural killer cell therapies can be manufactured from a variety of sources: these include peripheral blood, either steady-state or taking advantage of apheresis performed to collect hematopoietic stem and progenitor cells mobilized with growth factors such as granulocyte colony-stimulating factor (G-CSF), bone marrow, and cord blood.

As already described, NK cells are generally isolated through immune-selection techniques, using the canonical CD3⁻/CD56⁺ phenotype (42, 70), then cultured for functional activation and possibly expansion. Furthermore, NK cells can be genetically engineered to express natural or chimeric molecules empowering them with improved immune functions (5, 64).

Expansion and activation of potent cytotoxic NK cells require several signals for survival, proliferation, and activation. Culture conditions, thus, incorporate media and serum supplements, together with clinical-grade cytokines, monoclonal antibodies, or other soluble molecules, and possibly native or engineered cell feeders. Culture conditions can further be improved through the substitution of bioreactors to static conditions (Table 1).

As already mentioned, and since most protocols that use only cytokines result in limited NK cell expansion, the introduction of feeder cells in the culture protocol has been extensively tested. Feeder cells provide additional stimulatory signals necessary for NK cell proliferation. Monocytes provide humoral signals and cell-to-cell contacts hence can serve as feeder cells (71); irradiated autologous PBMC have been used as feeder cells to produce sufficient numbers of NK cells with acceptable purity (6, 72, 73). Alternatively, irradiated allogeneic cells have been evaluated: Epstein–Barr virus-transformed lymphoblastoid B cell lines (EBV-TM-LCL), K562 cells (leukemic cell line) engineered to express a membrane-bound form of IL-15 fused to the T-cell receptor CD8α and the 41BB ligand (74–78), or K562 cells transduced with IL-21 (79). Such feeder cells proved effective in preclinical validation of the production of clinically relevant numbers of NK cells, however, raise regulatory issues when it comes to manufacture medicinal products.

Since the presence of residual feeder cells in the final product is of major concern for clinical applications (80), alternative approaches are evaluated as substitutes. Anti-CD3 (OKT3) antibodies in addition to IL-2, with or without IL-15, produced substantial although lower fold expansion of CD3⁻/CD56⁺-enriched cells (81–86) than protocols that use feeder cells.

Expansion strategies of clinical-grade NK cells usually require 7–21 days of culture; up to 28 days of culture have been reported (87). There is an incentive to substitute animal or human serum-replete medium with animal and human component-free medium. The most commonly used media for CB-derived NK cells is glycostem basal growth medium (Clear Cell Technologies, Beernem, Belgium), preferred for being free of animal-derived components (52, 53). For PBMC-derived NK cells, preferred media are: X-Vivo™ serum-free media (Biowhittaker, Verviers, Belgium), AIM V^® serum-free medium (Thermo Fisher Scientific, Grand Island, NY, USA), Stem Cell Growth medium (Cell Genix, Freiburg, Germany), or complete Roswell Park Memorial Institute 1640 (Biowhittaker, Verviers, Belgium).

Media supplements still being used by some groups include GMP-grade human AB serum, pooled human AB plasma, or fetal bovine serum (FBS). GMP-grade cytokines (recombinant human IL-2 and IL-15), antibodies (anti-CD3-OKT3), and other ancillary reagents (nicotinamide-NAM) commonly serve as medium supplements for PBMC-derived NK cell generation. Additional growth factors and cytokines are necessary for CB-derived NK cells since the starting material is commonly CD34⁺ stem cells [stem cell factor (SCF), IL-7, IL-15, IL-2, IL-6, flt3 ligand (Flt3-L), thrombopoietin (TPO), G-CSF, low molecular weight heparin (LMWH), granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), MIP1α].

In addition to using T75 cell culture flasks, several groups have used culture bags (Baxter LifeCell^® or VueLife^®) (76, 83). On larger scales, gas-permeable static cell culture (G-Rex^®) flasks (Wilson Wolf Manufacturing, New Brighton, MN, USA) (78) or WAVE Bioreactor™ (GE Healthcare Life Sciences, Chicago, IL, USA) (83) served as expansion platforms.