Cellular immunotherapy is on the forefront of progress in cancer research. The success of CAR T cells has impacted the development of novel alternative cell anticancer strategies. NK cells possess similar functions to T effector cells but with decreased toxicity. Early studies had demonstrated the feasibility and safety of adoptive transfer of allogeneic NK cells [12–14]. Clinical response rates in clinical studies were variable: the therapeutic response depended on the diagnosis, stage of disease, timing of immunotherapy, NK alloreactivity, cell dose and frequency of infusions [12, 13, 15, 16].
Currently, the greatest rate of NK cell expansion from PBMCs (over mean 47,000-fold in a period of 3 weeks in in vitro experiments) has been achieved with K562-based APCs (Clone 9.mbIL21) that coexpress CD64/FcgRI, CD86/B7-2, CD137L/4-1BBL, truncated CD19, and membrane-bound IL-21 [9]. This feeder line is widely used and proved very effective in propagating NK cells from peripheral blood or cord blood for preclinical investigations [8, 17, 18] and clinical trials [16, 19, 20].
We have previously reported the creation of our own K-562-based cell line transduced to express 4-1BBL and mbIL-21 [10]. In our small-volume experiments, the median fold expansion of NK cells at 3 weeks was > 21,000 (range 3,000–300,000). Other groups have reported > 10,000 fold expansion of NK cells by using a feeder cell line based on the myeloid OCI-AML3 cell line (at 5 weeks) [21] and based on the B-lymphoblastoid 721.221 cell line (at 3 weeks) [22] transduced to express mbIL-21.
When comparing feeder lines based on mbIL-15 and mbIL-21, mbIL21 supports greater proliferation of NK cells than mbIL15 [9, 10, 23]. Thus, expansion of NK cells with feeder cells expressing mbIL-21 creates the possibility to generate substantially more NK cells from a single withdrawal of peripheral blood. In our study, at the end of culturing the median fold expansion of NK cells was 224.7 (range 42–647) with minimal expansion of total T-cells – 1.7 (range 0.4–9.2) – which were predominantly CD3 + CD56 + cells. If we were to use all our obtained cells for our patients as a single infusion, the NK cell dose would be 111 × 106 (range 18–379 × 106) cells/ kg, and the T cell dose would be 2.6 × 106 (range 0.6–13.7 × 106) cells/kg. Fold order of NK cells expansion depends on cell source, culture systems for cells propagation (flasks, G-Rex, rocking bioreactors or automated closed systems), number of restimulation with APCs, cytokines and way to calculate the fold of expansion. For instance, NK expansion efficiency varied from tens [16] to thousands [20] fold even when the same APCs line (Clone 9. mbIL21) was used.
An important consideration for clinical application of any cell product is the safety profile. The main concern of adoptive transfer of allogeneic NK cell products is T cell contamination and graft versus host disease (GVHD) as a consequence. Therefore, elimination of CD3 + cells before or after expansion is a key step in many protocols. This step is especially important when NK expansion is based on stimulation with high doses of cytokines (IL-2, IL-15 or IL-21) that also stimulate proliferation of T cells. However, when expanding NK cells by using a feeder line, low doses of IL-2 (10–100 IU/ml) are usually used [9, 24–26]. We used 50 IU/ml of IL-2 and the median purity of NK cells in the final cell products was 96.6%, despite the low initial content of NK cells and expansion without prior CD3 depletion. Importantly, GVHD has not been reported in the vast majority of studies utilising allogenic feeder-based expanded NK cells.
Therefore, we can conclude that application of genetically modified feeder cells for NK cell expansion from whole blood without leukapheresis and magnetic selection represents an opportunity to obtain an NK cell product with good expansion, high NK purity and low production cost. The cost is an especially important factor that delays the development and application of cell therapy technologies in countries with limited financial resources. However, the prospect of using serum-free media and G-Rex flasks would allow obtaining more safe NK cells in a shorter time with minimal manipulations. According to our results, the median duration of NK expansion was 18 days (range 14–25); using G-Rex would shorten the expansion period to 10–12 days.
In previous studies, NK cells expanded by using gene-modified feeder lines have shown high expression of activating receptors as well as in vitro antitumor cytotoxicity against different tumour cell lines [9, 21, 23]. We also demonstrated increased expression of the activating receptors NKp30, NKp44, NKp46 and NKG2D, as well as CD69, HLA-DR and CD96. In addition, NK cells acquired a less differentiated phenotype. After expansion, almost all NK cells were CD56bright, expressed NKG2A and did not express CD57. CD57 is a marker of mature, terminally differentiated NK cells with high cytotoxicity but decreased proliferative activity [27]. However, it has been shown that in specific conditions of cell stimulation, CD57 + human NK cells could acquire the CD57- phenotype. Using mbIL-21 for NK cell stimulation decreased CD57 expression [21, 28]. Besides, at the end of culturing the majority of NK cells expressed CD16; indeed, > 75% of NK cells had the double-bright (CD56bright/CD16bright) phenotype. It has been shown in previous studies that stimulating NK cells with a combination of cytokines (IL-2, IL-12 and IL-15), as well as mbIL-21- or mbIL-15-expressing feeder lines, produces CD16 + CD56bright NK cells with high cytotoxicity [9, 29, 30].
NK cell expansion resulted in increased expression of immune checkpoints LAG-3 and TIM-3, which are commonly associated with T cell exhaustion. The molecular mechanisms underlying NK cell exhaustion are not defined as clearly as they are for T cells. Unlike T cells, TIM-3 and TIGIT are expressed at high levels on unstimulated and functional NK cells [31]. A better understanding of the role of immune checkpoints for NK effector function would be beneficial to design immunotherapeutic approaches for cancer patients.
Despite the expression of exhaustion markers, the expanded NK cells were functionally active. They exhibited high cytolytic activity against leukaemia cell lines, high degranulation activity and production of cytokines after stimulation with K-562 cells. However, it is worth noting that expanded NK cells were less active in the tests performed against primary recipient blasts. Low cytotoxic NK cell activity against recipient blast cells might be explained by various peculiarities of antigen expression on blast cells (e.g. MIC A/B, ULBP1-6 or HLA class I molecules) but not by dysfunction of NK cells. Thus, expanded NK cells are capable of both IFNγ production and cytotoxicity and have phenotypic characteristics associated with both mature and immature NK cells. Our results support the suggestion about uncoupling phenotypic markers of maturation and canonical functions of NK cell subsets in ex vivo–expanded NK cells [30].