In an ovarian cancer xenograft model, NK-CAR-iPSC-NK cells displayed better anti-tumor activity when compared to PB-NK, iPSC-NK, and T-CAR-expressing-iPSC-NK cells and had greater circulation at day 10. immortalized NK cell lines require irradiation and are dependent on systemic IL-2 administration, which has been associated with adverse effects. In contrast, NK cells differentiated from induced pluripotent stem cells (iPSC-NK cells) offer an off-the-shelf alternative that may overcome Nisoxetine hydrochloride these bottlenecks. The development of a serum-free and feeder-free differentiation protocol allows for the manufacturing of clinically adaptable iPSC-NK cells that are equally as effective as primary NK cells and the NK-92 cell line for many indications. Moreover, genetic modifications targeting NK-mediated antibody-dependent cellular cytotoxicity capabilities, cytotoxicity, and checkpoint inhibitors may increase the therapeutic potential of iPSC-NK products. This review will highlight the current sources for NK therapies and their respective constraints, discuss recent developments in the manufacturing and genetic engineering of iPSC-NK cells, and provide an overview of ongoing clinical trials using NK cells. gene One of the issues associated with loss of cytotoxicity is the cleavage and shedding of the CD16 receptor. Upon NK activation, CD16 undergoes cleavage by the ADAM17 protease and is shed from the membrane, causing the NK cell to lose its ability to perform ADCC [121]. To circumvent this issue, Jing et al. transduced a mutated version of the CD16a receptor (CD16a/S197P) into iPSCs using a transposon [121]. A site-directed mutagenesis of the CD16a receptor (S197P) prevented cleavage by ADAM17 and resulted in stable expression of CD16 even upon activation by the K562 cell line. In a separate abstract, Blum et al. reported that the CD16a/S197P-transduced iPSC-NK cells were 97.5% CD16+ before stimulation and 95.2% CD16+ after stimulation [122]. They also reported that CD16a/S197P-transduced iPSC-NK also showed improved degranulation and better killing of the SKOV3 ovarian cancer Nisoxetine hydrochloride cell line when compared to unmodified iPSC-NK and PB-NK [122]. Studies incorporating CRISPR/Cas9 are also underway to determine the larger effects of deleting the Rabbit polyclonal to YY2.The YY1 transcription factor, also known as NF-E1 (human) and Delta or UCRBP (mouse) is ofinterest due to its diverse effects on a wide variety of target genes. YY1 is broadly expressed in awide range of cell types and contains four C-terminal zinc finger motifs of the Cys-Cys-His-Histype and an unusual set of structural motifs at its N-terminal. It binds to downstream elements inseveral vertebrate ribosomal protein genes, where it apparently acts positively to stimulatetranscription and can act either negatively or positively in the context of the immunoglobulin k 3enhancer and immunoglobulin heavy-chain E1 site as well as the P5 promoter of theadeno-associated virus. It thus appears that YY1 is a bifunctional protein, capable of functioning asan activator in some transcriptional control elements and a repressor in others. YY2, a ubiquitouslyexpressed homologue of YY1, can bind to and regulate some promoters known to be controlled byYY1. YY2 contains both transcriptional repression and activation functions, but its exact functionsare still unknown ADAM17 gene [122]. Another successful modification to prevent CD16 cleavage has been to transduce iPSCs with a recombinant Fc receptor with the extracellular region of CD64, and the intracellular and transmembrane region of CD16a (CD64/16A) using a transposon [124]. The CD64/16A receptor lacks the ADAM17 cleavage site, preventing CD16 downregulation upon NK activation. In an in vitro cytotoxicity assay, the transduced iPSC-NK cells exhibited greater ADCC against SKOV3 ovarian cancer cells when compared to untransduced iPSC-NK cells. Fate Therapeutics is conducting an analogous line of research with their iPSC-NK products, FT516 and FT538 (see Supplementary Table?2, Additional file?2). FT516 is an iPSC-NK product that has been engineered Nisoxetine hydrochloride with a high-affinity, non-cleavable CD16 (hnCD16) receptor at the iPSC stage to enhance ADCC and anti-tumor capabilities, and is undergoing phase I clinical trials in adults with hematologic malignancies (see Supplementary Table?2, Additional file?2, “type”:”clinical-trial”,”attrs”:”text”:”NCT04023071″,”term_id”:”NCT04023071″NCT04023071) [125]. A preclinical study reported that hnCD16-iPSC-NK cells displayed greater ADCC capabilities, CD107a expression, and IFN-gamma production compared to peripheral and unmodified iPSC-NK cells against various antibody-treated cancer cell lines [126]. While treatment with hnCD16-iPSC-NK, iPSC-NK, or PB-NK cells alone did not show a change in tumor burden, a combinatorial treatment of anti-CD20 and hnCD16-iPSC-NK showed a decrease in tumor burden 10?days after treatment in an in vivo B cell lymphoma mouse xenograft model. However, relapse occurred in most treated groups, but was rescued by multiple doses of treatment extending the mean survival from 52 to 76?days. The FT538 product also addresses issues with NK cytotoxicity, specifically in cases of multiple myeloma. Myeloma cells strongly express CD38 and are often treated with daratumumab, an anti-CD38 monoclonal antibody [127]. However, administration with daratumumab has been shown to demonstrate reductions in NK cell counts and activation due to fratricide from NK expression of CD38 [128]. To circumvent this, FT538, derived from a clonal master iPSC line, has been modified with a knockout of the CD38 receptor and knock-in of the hnCD16 receptor to increase ADCC and prevent exhaustion when used with anti-CD38 antibody treatments [129]. Nisoxetine hydrochloride Though useful strategies, completely preventing cleavage of CD16, could also be problematic since CD16 shedding has been established as a regulatory mechanism that sustains NK survival by assisting with detachment of NK cells from target cells [130]. Once a target cell is lysed, the NK cell detaches and continues its surveillance for other malignant cells and can normally kill up to seven target cells in a 12-h period [131]. However, Srpan et al. observed a 67% decrease in detachment of NK cells and found that NK cells stayed in contact with their target cells even after 8?h when modified with the mutant CD16 receptor (S197P) from the Jing et al. study [130]. While the.