Natural killer (NK) cells are increasingly considered as immunotherapeutic agents in particular in the fight against cancers. NK cell therapies are potentially broadly applicable and, different from their T cell counterparts, do not cause graft-versus-host disease. Efficacy and clinical in vitro or in vivo expansion of primary NK cells will however always remain variable due to individual differences of donors or patients. Long-term storage of clinical NK cell lots to allow repeated clinical applications remains an additional challenge. In contrast, the established and well-characterized cell line NK-92 can be easily and reproducibly expanded from a good manufacturing practice (GMP)-compliant cryopreserved master cell bank. Moreover, no cost-intensive cell purification methods are required. To date, NK-92 has been intensively studied. The cells displayed superior cytotoxicity against a number of tumor types tested, which was confirmed in preclinical mouse studies. Subsequent clinical testing demonstrated safety of NK-92 infusions even at high doses. Despite the phase I nature of the trials conducted so far, some efficacy was noted, particularly against lung tumors. Furthermore, to overcome tumor resistance and for specific targeting, NK-92 has been engineered to express a number of different chimeric antigen receptors (CARs), including targeting, for example, CD19 or CD20 (anti-B cell malignancies), CD38 (anti-myeloma) or human epidermal growth factor receptor 2 (HER2; ErbB2; anti-epithelial cancers). The concept of an NK cell line as an allogeneic cell therapeutic produced 'off-the-shelf' on demand holds great promise for the development of effective treatments.

Cyclosporine (CsA) nephrotoxicity is a severe complication in organ transplantation because it leads to impaired renal function and chronic allograft nephropathy, which is a major predictor of graft loss. Animal models and in vivo studies indicate that the transmembrane efflux pump P-glycoprotein contributes substantially to CsA nephrotoxicity. It was hypothesized that the TT genotype at the ABCB1 3435C3 T polymorphism, which is associated with decreased expression of P-glycoprotein in renal tissue, is a risk factor for developing CsA nephrotoxicity. In a case-control study, 18 of 97 patients developed CsA nephrotoxicity and showed complete recovery of renal function in all cases when switched to a calcineurin inhibitor-free regimen. Both recipients and donors were genotyped for ABCB1 polymorphisms at the positions 3435C3 T and 2677G3 T/A. For controlling for population stratification, two additional polymorphisms, CYP2D6*4 and CYP3A5*3, with intermediate allelic frequencies were studied. The P-glycoprotein low expressor genotype 3435TT only of renal organ donors but not of the recipients was overrepresented in patients with CsA nephrotoxicity as compared with patients without toxicity ( 2 ‫؍‬ 10.5; P ‫؍‬ 0.005). CsA dosage, trough levels, and the concentration per dose ratio were not different between the patient groups. In a multivariate model that included several other nongenetic covariates, only the donor's ABCB1 3435TT genotype was strongly associated with CsA nephrotoxicity (odds ratio, 13.4; 95% confidence interval, 1.2 to 148; P ‫؍‬ 0.034). A dominant role of the donor's ABCB1 genotype was identified for development of CsA nephrotoxicity. This suggests that P-glycoprotein is an important factor in CsA nephrotoxicity.

Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 108 CD56+CD3− cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56+CD3− NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.

Natural killer (NK) cells have been used in several clinical trials as adaptive immunotherapy. The low numbers of these cells in peripheral blood mononuclear cells (PBMC) have resulted in various approaches to preferentially expand primary NK cells from PBMC. While some clinical trials have used the addition of interleukin 2 (IL-2) to co-stimulate the expansion of purified NK cells from allogeneic donors, recent studies have shown promising results in achieving in vitro expansion of NK cells to large numbers for adoptive immunotherapy. NK cell expansion requires multiple cell signals for survival, proliferation and activation. Thus, expansion strategies have been focused either to substitute these factors using autologous feeder cells or to use genetically modified allogeneic feeder cells. Recent developments in the clinical use of genetically modified NK cell lines with chimeric antigen receptors, the development of expansion protocols for the clinical use of NK cell from human embryonic stem cells and induced pluripotent stem cells are challenging improvements for NK cell-based immunotherapy. Transfer of several of these protocols to clinical-grade production of NK cells necessitates adaptation of good manufacturing practice conditions, and the development of freezing conditions to establish NK cell stocks will require some effort and, however, should enhance the therapeutic options of NK cells in clinical medicine.

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