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CAR-NK Cell Therapy Flow Cytometry Assay

Posted on: March 04, 2021

What is the Primary Role of Natural killer (NK) cells?  

Natural killer (NK) cells are the predominant innate immune cells that mediate anti-tumor and anti-viral responses, and therefore possess good clinical utilization (Abel et al. 2018). Natural killer cells comprise 10–15% of peripheral blood lymphocytes and classically display a half-life of approximately 7–10 days in the circulation (Moretta et al. 2000).

NK cells show spontaneous cytolytic activity against tumors and viral infections. NK cells also secrete different cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), and a range of chemokines (CCL1, CCL2, CCL3, CCL4, CCL5, and CXCL8) (Paul and Lal 2017).

Typically using flow cytometry, NK cells are identified as CD16+ (FcƔ receptor III), and CD56+ and their lack of T cell receptor (TCR) and CD3 expression. Two distinct subpopulations are readily identified: CD56bright and CD56dim, and these subpopulations differ in their cytotoxic potential and cytokine production. It appears that CD56bright NK cells are the precursor cells of the CD56dim subset, although both subtypes play critical roles in immune responses.

Preclinical and early clinical trials with NK-cell immunotherapies exhibit safety and encouraging clinical outcomes against hematologic malignancies. Developing off-the-shelf and vital cytotoxic NK cells is necessary for clinical applications. NK and T cells use distinct tumor cell recognition mechanisms. They can be sensitive or synergistic to different tumor types. So, the clinical outcome of combination therapy with NK and T cells needs to be confirmed with large-scale clinical trials. Allogenic NK cells from peripheral blood are safe and satisfactorily effective against tumors (Fang et al. 2018). 

Transfection efficiency for primary NK cells particularly remains the bottleneck. Transiently inhibiting the anti-viral defense signaling pathway leads to remarkably increased virus transduction efficiency, but it is not practical for the large-scale manufacture of CAR-NK cells (Stulu et al. 2012).  An efficient, reliable, and convenient transfection protocol is the bottleneck for developing gene-modified NK cells, an overcoming these technical barriers will be key to the expansion of these systems for clinical applications.

CAR -T Cell Therapy

T lymphocytes are engineered with synthetic receptors known as chimeric antigen receptors (CAR) in CART Cell therapy. The CAR-T cell is an effector T cell that recognizes and eliminates specific cancer cells, independent of major histocompatibility complex molecules. (Zhai et al. 2018). Chimeric antigen receptors (CARs) cells have recombinant receptor constructs expressed in T cells to target cells expressing specific antigens.

CAR-T therapy was developed with the construction of recombinant TcRs that replaced the TcR V regions with antigen-specific antibody V regions. The chimeric TcRs retained the normal extracellular C region, the transmembrane segment, and the cytoplasmic signaling domains, and therefore maintained the ability to induce T-cell proliferation, interleukin production, and cell lysis. Furthermore, these chimeric TcRs were non-MHC-restricted and universal in the sense that a given chimeric construct could be transfected into T-cells from any individual.  T cells expressing CAR have been shown to recognize a wide range of surface antigens, including glycolipids, carbohydrate moieties, and proteins (Morello et. al., 2016), and can attack malignant cells expressing these antigens. 

CAR-T cell approaches have revolutionized the landscape of cancer therapeutics, particularly against hematological malignancies, and remain the most promising approach for treating many types of cancers.

CAR-NK Cell Therapy 

CAR-NK cell therapy has proven to kill hematological and solid tumor cells in preclinical and clinical trials, demonstrating its potential as an off-the-shelf product with broad clinical applications (Siegler et al., 2018) while eliminating the risk of Graft Versus Host Disease (GVhD) associate with CAR-T cells. NK92 cell line is usually used in current CAR-NK clinical trials. 

CAR-NK cells eliminate tumors not only through CAR's ability to specifically recognize antigen-expressing tumors but also through NK cell receptors themselves. It is the balance of stimulatory and inhibitory signals, not antigen specificity, that determines NK cell activity (Davies 2014). 

CAR-engineered NK (CAR-NK) cells have great potential for cancer therapy. There is a bottleneck in the development of CAR-NK cell therapies. CARs were designed for building CAR-T cells and, as such, are a suboptimal choice for application to NK cells. The location of CAR binding epitopes and their distance from the CAR-NK cell surface affects their ability to bind antigens and activates CAR-NK cells (Zhang et al., 2017).

With more clinical data provided in the next few years, CAR-NK cell therapies may lead to revolutionary advances in tumor immunotherapy.

CAR NK cell therapy has a strong potential to control tumor growth, showing resistance to conventional immunotherapy. CAR-NK therapy has significant advantages over CART therapy, such as (1) better safety, (2) multiple mechanisms for activating cytotoxic activity, and (3) high feasibility for ‘off-the-shelf’ manufacturing. CAR-NK cells could be engineered to target diverse antigens and ultimately achieve an effective anti-tumor response. 

CAR-NK Cell therapy sample analysis by Flow Cytometry. 

Several flow cytometry-based assays are developed for CAR-NK cells. The use of protein transport inhibitors (e.g., brefeldin A and monensin) and cell permeabilization methods in combination with conventional surface staining protocols have enabled scientists to study chemokine and cytokine production in different specific lymphocyte subsets (e.g., T, B or NK cells) (Jack et al 2015). Moreover, different flow cytometry-based assays have been developed to monitor T and NK cell cytotoxicity (Sarah et al 2016).

Flow Cytometry assay at Flowmetric Inc. 

Incorporating into the CAR-NK manufacturing process are several Quality Checkpoints. Rigorous QC testing is performed in batch release tests for T-cell viability and sample purity, T-cell potency in the form of effector function and activation, and microbiological safety. 

The robustness of the product quality control process is demonstrated through quality control of the CAR-NK product and the preventative/corrective action, change control, and review protocols in place. Release testing frequently involves flow cytometry analysis of the cells to determine the purity of the therapeutic preparation and potency testing.

CAR-NK therapies need further development, and several challenges need to be solved. NK expansion methods are more diverse as compared to T. cells. It needs to be shown that current CAR-NK therapy methods will be suitable for large scale clinical and commercial production.  Continuous improvement will be required to achieve consistent CAR-NK products. Further exploration of NK cells to control tumor growth is needed and how best to select, characterize, and culture NK cell subsets with the most excellent anti-tumor activity (Romee et al. 2016).

sibtain-ahmed

Authored by: Dr. Sibtain Ahmed

 

 

 

Dr. Sibtain Ahmed is a scientific writer and a member of FlowMetric’s Business Development teamSibtain is a skilled a biochemist with experience in the field of biologics, and cell and gene therapy manufacturing, drug discovery, vaccines, and fermentation. Sibtain earned his B.S. in Biology at the University of the Punjab and his Ph.D. from the University of Agriculture Faisalabad. Sibtain did postdoc research at the University of New Mexico and the University of California San Diego. Sibtain’s previous work history includes working at Thermo Fisher Scientific, Hologic, and the Genomics Institute of the Novartis Research Foundation.  Sibtain has authored peer-reviewed articles/book chapters, presented posters, and has given oral talks in
scientific meetings.

References.

Alex M Abel , Chao Yang , Monica S Thakar , Subramaniam Malarkannan . 2018. Natural Killer Cells: Development, Maturation, and Clinical Utilization2018. 13; 9:1869. doi: 10.3389/fimmu.2018.01869. 

Sourav Paul Girdhari Lal. 2017. The Molecular Mechanism of Natural Killer Cells Function and Its Importance in Cancer Immunotherapy. Front Immunol. 13; 8:1124. doi: 10.3389/fimmu.2017.01124. 

 Moretta A, Bottino C, Mingari MC, Biassoni R, Moretta L. What is a natural killer cell? Nat Immunol (2002) 3(1):6–810.1038/ni0102-6

Fang Fang , Weihua Xiao , Zhigang Tian. 2018. Challenges of NK cell-based immunotherapy in the new era. Front Med. 12(4):440-450. doi: 10.1007/s11684-018-0653-9.

Sutlu T, Nyström S, Gilljam M, Stellan B, Applequist SE, Alici E. Inhibition of intracellular anti-viral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy. Hum Gene The. 2012; 23(10): 1090–1100

Zijun Zhao , Yu Chen , Ngiambudulu M Francisco , Yuanqing Zhang , Minhao Wu. 2018. The application of CAR-T cell therapy in hematological malignancies: advantages and challenges. Acta Pharm Sin B. 8(4):539-551. doi: 10.1016/j.apsb.2018.03.001. 

Morello A, Sadelain M, Adusumilli P. (2016) Mesothelin-targeted CARs: driving T cells to solid tumors. Cancer Discov. 6:133–46

  1. Zhang, P. Oberoi, S. Oelsner, et al.Chimeric antigen receptor-engineered NK-92 cells: an off-the-shelf cellular therapeutic for targeted elimination of cancer cells and induction of protective anti-tumor immunity. Front. Immunol., 8 (2017), p. 533

J.O.J. Davies, K. Stringaris, A.J. Barrett, et al. Opportunities and limitations of natural killer cells as adoptive therapy for malignant disease. Cytotherapy, 16 (2014), pp. 1453-1466

E.L. Siegler, Y. Zhu, P. Wang, et al. Off-the-shelf CAR-NK cells for cancer immunotherapy

Cell Stem Cell, 23 (2018), pp. 160-161

Romee R, Rosario M, Berrien-Elliott MM et al. Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia. Sci. Trans. Med. 2016; 8(357): 357ra123.

Jacobs B, et al. NK Cell Subgroups, Phenotype, and Functions After Autologous Stem Cell Transplantation. Front. Immunol. 2015;6:583. 

Sara TognarelliBenedikt JacobsNina Staiger, and Evelyn Ullrich. 2016. Flow Cytometry-based Assay for the Monitoring of NK Cell Functions. J Vis Exp. 2016; (116): 54615.