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.
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.
Autologous blood doping (ABD) refers to the transfusion of one's own blood after it has been stored. Although its application is prohibited in sports, it is assumed that ABD is applied by a variety ...of athletes because of its benefits on exercise performance and the fact that it is not detectable so far. Therefore, this study aims at identifying changes in hematological and hemorheological parameters during the whole course of ABD procedure and to relate those changes to exercise performance. Eight healthy men conducted a 31-week ABD protocol including two blood donations and the transfusion of their own stored RBC volume corresponding to 7.7% of total blood volume. Longitudinal blood and rheological parameter measurements and analyses of RBC membrane proteins and electrolyte levels were performed. Thereby, responses of RBC sub-populations-young to old RBC-were detected. Finally, exercise tests were carried out before and after transfusion. Results indicate a higher percentage of young RBC, altered RBC deformability and electrolyte concentration due to ABD. In contrast, RBC membrane proteins remained unaffected. Running economy improved after blood transfusion. Thus, close analysis of RBC variables related to ABD detection seems feasible but should be verified in further more-detailed studies.
Objective Interleukin (IL)-15 is a promising novel cytokine for natural killer (NK) cell activation and survival. We studied the effects of IL-15 compared to IL-2 on NK cells in long-term cultures ...for clinical translation. Materials and Methods CD56+ CD3− NK cells were expanded with IL-2 or IL-15 for 2 to 4 weeks within lymphokine-activated killer (LAK) cell cultures (LAK-NK) in serum-enriched AIM V or CellGro Stem Cell Growth Medium (SCGM). Cell growth, viability, and NK cell content were monitored and cytotoxicity assessed in a flow cytometric cytotoxicity assay. Results IL-15 (100–1000 U/mL) could replace IL-2 (1000 U/mL) in AIM V cultures to achieve efficient LAK cell expansion. However, IL-15–stimulated LAK cells exceeded cytotoxicity of IL-2–stimulated LAK cells against K562, notably at later culture points. In the powerful CellGro SCGM, LAK cells expanded over 28 days an average of 905-fold ± 320-fold standard error of the mean (SEM) for IL-2 (500 U/mL) and 484-fold ± 98-fold SEM for IL-15 (500 U/mL), and NK cells within such LAK cultures expanded an average of 2320-fold ± 975-fold SEM for IL-2 and 1084-fold ± 309-fold SEM for IL-15. Importantly, such IL-15–activated LAK-NK cells retained enhanced cytotoxicity at later culture points against K562 as well. IL-15–stimulated effectors were also highly cytotoxic against hematological targets MOLT-4 and KU812 and nontoxic against autologous nonmalignant cells. Interestingly, IL-15–LAK-NK cells showed overall significant upregulation of the main activating and inhibitory NK cell receptors after long-term cytokine stimulation. Conclusions Our results demonstrate the potential for IL-15 to support large-scale expansion of clinical-grade LAK-NK effectors, which could retain enhanced longer-term potency and preserve activation receptors in therapy of hematological malignancies. Protocols are readily clinically translatable.
Natural killer (NK) cell therapies are emerging worldwide as promising anticancer treatments, exploiting the fast cytolytic action of NK effectors and their potentially broad applicability against a ...wide range of malignancies. Until recently, clinical protocols have mainly involved freshly isolated NK cells or short-term activated NK cells or lymphokine-activated killer (LAK) cells. However, overall effector numbers and their anticancer potencies remained restricted, which poses a limiting factor to clinical efficacy. Recent developments in the field aim to improve clinical trial designs by increasing effector to target cell ratios in vivo and by application of superior cytotoxic NK effectors. Large-scale production of clinical grade NK cells through long-term activation in ex vivo cultures are another novel means in achieving these goals. However, such procedures require compliance with the strict Good Manufacturing Practice (GMP) regulations to ensure quality and safety of the NK cell product. Although the overall number of new protocols still remains comparably low, some of the protocols are already translated into clinical use. Also striking is the diversity of the different protocols proposed. We highlight in this review the most recent developments in the NK cell field with a focus on long-term NK cell expansion. Critical issues relating to this novel and promising type of therapy are highlighted and discussed.
The major mechanism for NK cell lysis of tumor cells is granule-mediated cytotoxicity. Polarization of granules is a prelude to the release of their cytotoxic contents in response to target-cell ...binding. We describe the novel observation of constitutive granule polarization in the cytotoxic NK cell line, KHYG-1. Continuous degranulation of KHYG-1 cells, however, does not occur and still requires target-cell contact. Disruption of microtubules with colcemid is sufficient to disperse the granules in KHYG-1 and significantly decreases cytotoxicity. A similar effect is not obtained by inhibiting extracellular signal-related kinase 2 (ERK2), the most distal kinase investigated in the cytolytic pathway. Disruption of microtubules significantly down-regulates activation receptors, NKp44 and NKG2D, implicating them as potential microtubule-trafficking receptors. Such changes in upstream receptor expression may have caused deactivation of ERK2, since NKG2D cross-linking also leads to receptor down-regulation and diminished ERK phosphorylation. Thus, a functional role for NKG2D in KHYG-1 cytotoxicity is demonstrated. Moreover, the novel primed state may contribute to the high cytotoxicity exhibited by KHYG-1.
Strategies are emerging to apply natural killer (NK) cells as therapeutic agents against a broad range of malignancies. Novel clinical approaches aim to overcome limitations of original therapies, ...which have utilized lymphokine activated killer cells or systemic cytokine treatments. Remarkable results, including survival improvements and amelioration of graft versus host disease, were obtained with alloreactive NK cells in some cases. Other approaches in clinical evaluation include targeting heat-shock protein (Hsp) 70 expressing tumors with pre-stimulated autologous NK cells or the application of an NK cell line, NK-92, with enhanced cytolytic activity. Further mechanistic insights into NK cell cytotoxicity are a prelude to improved clinical cancer therapies.
Clinical application of natural killer (NK) cells against leukemia is an area of intense investigation. In human leukocyte antigen-mismatched allogeneic hematopoietic stem cell transplantations ...(HSCT), alloreactive NK cells exert powerful anti-leukemic activity in preventing relapse in the absence of graft-versus-host disease, particularly in acute myeloid leukemia patients. Adoptive transfer of donor NK cells post-HSCT or in non-transplant scenarios may be superior to the currently widely used unmanipulated donor lymphocyte infusion. This concept could be further improved through transfusion of activated NK cells. Significant progress has been made in good manufacturing practice (GMP)-compliant large-scale production of stimulated effectors. However, inherent limitations remain. These include differing yields and compositions of the end-product due to donor variability and inefficient means for cryopreservation. Moreover, the impact of the various novel activation strategies on NK cell biology and in vivo behavior are barely understood. In contrast, reproduction of the third-party NK-92 drug from a cryostored GMP-compliant master cell bank is straightforward and efficient. Safety for the application of this highly cytotoxic cell line was demonstrated in first clinical trials. This novel ‘off-the-shelf' product could become a treatment option for a broad patient population. For specific tumor targeting chimeric-antigen-receptor-engineered NK-92 cells have been designed.
Resting normal human B cells express negligible c-src mRNA or Src protein tyrosine kinase; however, upon induction of proliferation, these cells express high levels of both mRNA and protein and show ...a concomitant increase in tyrosine kinase activity of immunoprecipitated Src. Src expression was most pronounced upon stimulation with CD154, and to a lesser extent CD70, Staphylococcus aureus, Cowan strain I and phorbol ester, and correlated with the activation of the cells. Transfection of cDNA for human wild-type or kinase-dead Src into Raji B cells resulted in an increase and decrease, respectively, of the cell numbers in culture, showing a direct correlation of proliferation to the expression of Src that was corroborated using anti-sense oligodeoxynucleotides and chemical inhibitors. Furthermore, the human B cell lines, Namalwa, Daudi and Raji express low levels of Src but express very high levels of Src after stimulation with CD154 that showed a correlation with increased activation. This is the first report of Src detectable in normal B cells. The finding that Src expression is inducible and correlates with stimulation by CD154 and the proliferation of the B cells suggests that Src may play a specific role in normal and transformed B cell activation/proliferation pathways mediated primarily through CD40 stimulation.
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