Qualification of a flow cytometry-based method for the evaluation of in vitro cytotoxicity of GTA002 natural killer cell therapy.

Background: Natural Killer (NK) cell-based therapies represent a ground-breaking opportunity for the treatment of solid tumors and hematological malignancies. NK cell manufacturing under good manufacturing practice (GMP) is complex and requires attentive assessment the product's safety and efficacy through quality control (QC). Release testing includes monitoring of in vitro cell expansion, differentiation, purity, phenotype, and cytotoxicity. As NK cells are biologically active products, the establishment of potency methods is particularly relevant; surrogate or improper assays can lead to rejection of qualifiable batches or to release of products that falsely meet potency specifications, potentially causing low efficacy during clinical trials. As cell-based therapeutics are highly heterogeneous, no universal guidelines for product characterization are available, and developers must invest significant effort in establishing and validating robust and fit-to-purpose assays. In this study, we describe the qualification procedure of a flow cytometry-based analytical method to assess in vitro potency of GTA002 NK cells, to be applied to oNKord®/inaleucel allogeneic off-the-shelf NK cell product from Glycostem Therapeutics, undergoing a Phase I/IIa clinical trial in acute myeloid leukemia (AML) patients (NCT04632316). Methods: First, we established multi-color flow cytometry panels to quantitatively determine the count of effector (E) GTA002 cells and leukemia target (T) K562 cells alone and in co-culture at different E:T ratios (10:1, 3:1, 1:1). Effector potency was then qualitatively expressed as percentage of cytotoxicity. Next, we defined protocols for method qualification to assess the pivotal features of the assays, including accuracy, precision, linearity, range, specificity, robustness, and carryover; quantitative acceptance criteria were determined for all parameters. Results of the qualification procedure are reported and discussed against pre-defined acceptance criteria. Results: Overall, our methods show robust performance across all parameters, ensuring QC-compliant assessment of NK cell potency as part of the release test panel for clinical batches. Notably, we identified relevant aspects to address when progressing towards method validation to support pivotal clinical studies. Conclusions: This article provides a "case-study" of how analytical method development for cell therapeutics is planned and executed from early clinical stages, anticipating the need to establish robust procedures to overcome scientific and regulatory challenges during method validation.

Lipid nanoparticle-mediated messenger RNA delivery for ex vivo engineering of natural killer cells.

Natural killer (NK) cells participate in the immune system by eliminating cancer and virally infected cells through germline-encoded surface receptors. Their independence from prior activation as well as their significantly lower toxicity have placed them in the spotlight as an alternative to T cells for adoptive cell therapy (ACT). Engineering NK cells with mRNA has shown great potential in ACT by enhancing their tumor targeting and cytotoxicity. However, mRNA transfection of NK cells is challenging, as the most common delivery methods, such as electroporation, show limitations. Therefore, an alternative non-viral delivery system that enables high mRNA transfection efficiency with preservation of the cell viability would be beneficial for the development of NK cell therapies. In this study, we investigated both polymeric and lipid nanoparticle (LNP) formulations for eGFP-mRNA delivery to NK cells, based on a dimethylethanolamine and diethylethanolamine polymeric library and on different ionizable lipids, respectively. The mRNA nanoparticles based on cationic polymers showed limited internalization by NK cells and low transfection efficiency. On the other hand, mRNA-LNP formulations were optimized by tailoring the lipid composition and the microfluidic parameters, resulting in a high transfection efficiency (∼100%) and high protein expression in NK cells. In conclusion, compared to polyplexes and electroporation, the optimized LNPs show a greater transfection efficiency and higher overall eGFP expression, when tested in NK (KHYG-1) and T (Jurkat) cell lines, and cord blood-derived NK cells. Thus, LNP-based mRNA delivery represents a promising strategy to further develop novel NK cell therapies.

Early TRAIL-engagement elicits potent multimodal targeting of melanoma by CD34+ progenitor cell-derived NK cells.

Umbilical cord blood (UCB) CD34+ progenitor cell-derived natural killer (NK) cells exert efficient cytotoxicity against various melanoma cell lines. Of interest, the relative cytotoxic performance of individual UCB donors was consistent throughout the melanoma panel and correlated with IFNγ, TNF, perforin and granzyme B levels. Importantly, intrinsic perforin and Granzyme B load predicts NK cell cytotoxic capacity. Exploring the mode of action revealed involvement of the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46 and most importantly of TRAIL. Strikingly, combinatorial receptor blocking led to more pronounced inhibition of cytotoxicity (up to 95%) than individual receptor blocking, especially in combination with TRAIL-blocking, suggesting synergistic cytotoxic NK cell activity via engagement of multiple receptors which was also confirmed in a spheroid model. Importantly, lack of NK cell-related gene signature in metastatic melanomas correlates with poor survival highlighting the clinical significance of NK cell therapies as a promising treatment for high-risk melanoma patients.

Natural killer cells in clinical development as non-engineered, engineered, and combination therapies.

Natural killer (NK) cells are unique immune effectors able to kill cancer cells by direct recognition of surface ligands, without prior sensitization. Allogeneic NK transfer is a highly valuable treatment option for cancer and has recently emerged with hundreds of clinical trials paving the way to finally achieve market authorization. Advantages of NK cell therapies include the use of allogenic cell sources, off-the-shelf availability, and no risk of graft-versus-host disease (GvHD). Allogeneic NK cell therapies have reached the clinical stage as ex vivo expanded and differentiated non-engineered cells, as chimeric antigen receptor (CAR)-engineered or CD16-engineered products, or as combination therapies with antibodies, priming agents, and other drugs. This review summarizes the recent clinical status of allogeneic NK cell-based therapies for the treatment of hematological and solid tumors, discussing the main characteristics of the different cell sources used for NK product development, their use in cell manufacturing processes, the engineering methods and strategies adopted for genetically modified products, and the chosen approaches for combination therapies. A comparative analysis between NK-based non-engineered, engineered, and combination therapies is presented, examining the choices made by product developers regarding the NK cell source and the targeted tumor indications, for both solid and hematological cancers. Clinical trial outcomes are discussed and, when available, assessed in comparison with preclinical data. Regulatory challenges for product approval are reviewed, highlighting the lack of specificity of requirements and standardization between products. Additionally, the competitive landscape and business field is presented. This review offers a comprehensive overview of the effort driven by biotech and pharmaceutical companies and by academic centers to bring NK cell therapies to pivotal clinical trial stages and to market authorization.

Adoptive NK Cell Therapy: A Promising Treatment Prospect for Metastatic Melanoma.

Adoptive cell therapy (ACT) represents a promising alternative approach for patients with treatment-resistant metastatic melanoma. Lately, tumor infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor (CAR)-T cell therapy have shown improved clinical outcome, compared to conventional chemotherapy or immunotherapy. Nevertheless, they are limited by immune escape of the tumor, cytokine release syndrome, and manufacturing challenges of autologous therapies. Conversely, the clinical use of Natural Killer (NK) cells has demonstrated a favorable clinical safety profile with minimal toxicities, providing an encouraging treatment alternative. Unlike T cells, NK cells are activated, amongst other mechanisms, by the downregulation of HLA class I molecules, thereby overcoming the hurdle of tumor immune escape. However, impairment of NK cell function has been observed in melanoma patients, resulting in deteriorated natural defense. To overcome this limitation, "activated" autologous or allogeneic NK cells have been infused into melanoma patients in early clinical trials, showing encouraging clinical benefit. Furthermore, as several NK cell-based therapeutics are being developed for different cancers, an emerging variety of approaches to increase migration and infiltration of adoptively transferred NK cells towards solid tumors is under preclinical investigation. These developments point to adoptive NK cell therapy as a highly promising treatment for metastatic melanoma in the future.

miR-146a Exerts Differential Effects on Melanoma Growth and Metastatization.

UNLABELLED: Malignant melanoma is the most aggressive form of skin cancer; therefore, it is crucial to disclose its underlying molecular mechanisms. MicroRNAs (miRNAs) are small endogenous noncoding RNAs able to posttranscriptionally downregulate the expression of direct target genes. Using a melanoma progression model, miR-146a was identified as a key double-acting player in melanoma malignancy. In fact, miR-146a is able to enhance tumor growth, while it suppresses dissemination. It was determined that miR-146a coordinated melanoma cell growth by its direct targets lunatic fringe (LFNG) and NUMB, which operate on the NOTCH/PTEN/Akt pathway; while inhibition of metastasis formation was linked to decreased expression of ITGAV and ROCK1. Relevantly, miR-146a expression correlated with melanoma recurrence and was enriched in both patient-derived melanoma and cutaneous metastasis specimens, while its direct targets were depleted. However, miR-146a levels drop in circulating tumor cells (CTCs), suggesting the necessity for miR-146a expression to fluctuate during tumor progression in order to favor tumor growth and allow dissemination. This study reconciles the contradictory biologic functions of miR-146a in melanoma progression and unravels distinct molecular mechanisms that need to be considered for therapeutic interventions. IMPLICATIONS: miR-146a controls melanoma progression in a dual way, promoting growth and inhibiting dissemination; however, it is poorly expressed in CTCs, resulting in overall tumor spreading and distant-site colonization. Mol Cancer Res; 14(6); 548-62. ©2016 AACR.

CyTRANSFINDER: a Cytoscape 3.3 plugin for three-component (TF, gene, miRNA) signal transduction pathway construction.

BACKGROUND: Biological research increasingly relies on network models to study complex phenomena. Signal Transduction Pathways are molecular circuits that model how cells receive, process, and respond to information from the environment providing snapshots of the overall cell dynamics. Most of the attempts to reconstruct signal transduction pathways are limited to single regulator networks including only genes/proteins. However, networks involving a single type of regulator and neglecting transcriptional and post-transcriptional regulations mediated by transcription factors and microRNAs, respectively, may not fully reveal the complex regulatory mechanisms of a cell. We observed a lack of computational instruments supporting explorative analysis on this type of three-component signal transduction pathways. RESULTS: We have developed CyTRANSFINDER, a new Cytoscape plugin able to infer three-component signal transduction pathways based on user defined regulatory patterns and including miRNAs, TFs and genes. Since CyTRANSFINDER has been designed to support exploratory analysis, it does not rely on expression data. To show the potential of the plugin we have applied it in a study of two miRNAs that are particularly relevant in human melanoma progression, miR-146a and miR-214. CONCLUSIONS: CyTRANSFINDER supports the reconstruction of small signal transduction pathways among groups of genes. Results obtained from its use in a real case study have been analyzed and validated through both literature data and preliminary wet-lab experiments, showing the potential of this tool when performing exploratory analysis.

MSC-regulated microRNAs converge on the transcription factor FOXP2 and promote breast cancer metastasis.

Mesenchymal stem/stromal cells (MSCs) are progenitor cells shown to participate in breast tumor stroma formation and to promote metastasis. Despite expanding knowledge of their contributions to breast malignancy, the underlying molecular responses of breast cancer cells (BCCs) to MSC influences remain incompletely understood. Here, we show that MSCs cause aberrant expression of microRNAs, which, led by microRNA-199a, provide BCCs with enhanced cancer stem cell (CSC) properties. We demonstrate that such MSC-deregulated microRNAs constitute a network that converges on and represses the expression of FOXP2, a forkhead transcription factor tightly associated with speech and language development. FOXP2 knockdown in BCCs was sufficient in promoting CSC propagation, tumor initiation, and metastasis. Importantly, elevated microRNA-199a and depressed FOXP2 expression levels are prominent features of malignant clinical breast cancer and are associated significantly with poor survival. Our results identify molecular determinants of cancer progression of potential utility in the prognosis and therapy of breast cancer.

A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance.

In this report, we have shown that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. MiR146b expression was significantly higher in the mammary glands of pregnant and lactating mice than in virgin mice. Furthermore, miR146b levels were significantly higher in mouse mammary glands exposed to the sex hormones, estrogen and progesterone, compared with those of untreated control animals. Pregnancy-derived primary mouse mammary epithelial cells in which miR146b was knocked down showed a significant reduction in the number of hollow acinar organoid structures formed on three-dimensional Matrigel and in ß-casein expression. This demonstrates that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. It has been shown that mouse mammary luminal progenitors give rise to hollow organoid structures, whereas solid organoid structures are derived from stem cells. Among several miR146b targets, miR146b knockdown resulted in preferential STAT3ß overexpression. In the primary mouse mammary epithelial cells, overexpression of STAT3ß isoform caused mammary epithelial cell death and a significant reduction in ß-casein mRNA expression. Therefore, we conclude that during pregnancy miR146b is involved in luminal alveolar progenitor cell maintenance, at least partially, by regulating STAT3ß.