CAR NK cell research workflow

Multiple pre-clinical studies during the last years have indicated a growing interest in CAR NK cell research and engineering (Kloess, S. et al. 2019; Knorr, D.A. et al. 2013; Liu, E. et al. 2020; Nowakowska, P. et al. 2018). 

With our advanced technologies for each workflow step, we at Miltenyi Biotec support CAR NK cell research and therapeutic development. Starting with enrichment of a well-defined NK cell population, we further offer optimized solutions for activation, transduction, expansion, and finally the analysis and phenotyping of engineered CAR NK cells.

Based on Miltenyi Biotec’s experience and expertise in NK cell manufacturing as well as CAR T and CAR NK cell research (Bari, R. et al. 2019), this straightforward, translational workflow leads through the complete CAR NK engineering procedure. Tailored solutions for each workflow step enable research and development of engineered NK cells in small-scale settings. 

Starting CAR NK cell engineering with a well-defined, enriched NK cell population is key to obtain robust, standardized, and reproducible results. Depending on the starting material – PBMCs or blood products – standard MACS® MicroBeads and Isolation Kits or the innovative StraightFrom® Technology are used to enrich viable NK cell populations. Furthermore, MACS MicroBeads in MACS GMP Quality enable a seamless transition into the clinical setting. 

Enrichment of CD56+ NK cells with MACS Technology

NK cell enrichment from PBMCs increases reproducibility 

Sequential isolation of NK cells using MACS MicroBeads results in a homogeneous starting cell population. First, magnetically labeled CD3+ T cells are depleted from PBMCs. Subsequently, CD56+ NK cells are enriched by positive selection.  

This two-step separation of NK cells minimizes donor variability, improves transduction efficiency, and avoids vector consumption by unwanted cells, allowing for robust, standardized, and reproducible results.  

If subsequent magnetic sorting steps are required, untouched cell isolation with the MACS NK Cell Isolation Kit allows for isolation of unlabeled target cells via depletion of non-target cells.

Efficient NK cell transduction starts with physiological NK cell activation to induce cell proliferation. In addition, cultivation in NK MACS Medium supplemented with human AB serum and MACS Cytokines allows for optimal cell expansion. To enable a smooth transition from research to clinical-grade CAR NK cell manufacturing, NK MACS Medium is also available in MACS GMP Quality. 

Superior expansion of isolated NK cells 

Highly efficient expansion of human NK cells

Comparison of NK cells either activated and cultured in NK MACS Medium supplemented with 5% AB serum and 500 UI/mL of IL-2, two competitor media, or TexMACS™ Medium developed for T cell expansion show superior expansion of NK cells in NK MACS Media. 

Transgene expression depends on gene transfer efficiency. But viral transduction of suspension cells with retroviral or lentiviral vectors is often inefficient and requires the use of transduction enhancers. Using the synthetic, non-toxic soluble Vectofusin®-1 with a simple and easy-to-follow protocol enhances the transduction efficiency of viral vectors.  

Standardized characterization of CAR NK cells is crucial to assess the success of CAR NK cell therapies. High-quality reagents, instruments, and software enable reliable analysis of phenotype, activation status, and functional profile. 

The recordings of Miltenyi Biotec´s Cell Therapy Analytics Day 2021 summarize lessons learned, best practices, and regulatory hurdles in cell manufacturing analytics. 

Phenotypic characterization

Check the phenotype of your engineered CAR NK cells with suitable assays, for example, to analyze the NK cell activation status and to detect expression of the CAR construct on the cell surface. 

Validated panels for NK cell flow analysis

Set-up and validation of multicolor flow cytometry experiments are tedious but crucial for successful phenotypic NK cell characterization. Miltenyi Biotec Tested Panels (MBTP) of fluorochrome-conjugated REAfinity™ Recombinant Antibodies are already optimized and validated. They combine antibody panels, step-by-step protocols, and gating strategies to streamline CAR NK cell flow analysis. 

Mechanism of  CAR NK cell detection
Mechanism of  CAR NK cell detection

Mechanism of CAR NK cell detection with CAR Detection Reagents

Ready-to-use, biotinylated CAR Detection Reagents were developed for the detection of cells that are engineered to express a specific CAR on their cell surface, e.g., CD19 CAR, CD22 CAR, or BCMA CAR. CAR-engineered cells are reliably identified and enumerated by background-free flow cytometry analysis, without any additional FcR blocking steps. Binding of the antigen-based fusion protein (CAR Detection Reagent) to CAR NK cells simulates in vivo binding of CAR NK cells to the tumor antigen.

Detection of viable transduced NK cells after expansion 

Reliable detection and enumeration of viable CAR-expressing NK cells

Flow cytometric analysis of CAR-engineered NK cells with CAR Detection Reagent (B) shows successful expression of the CAR molecule. 

Functional profiling of CAR NK cells

Functionality of engineered NK cells is assessed by various assays. Our flow cytometry solutions allow for analysis of antigen-specific target cell killing and intracellular as well as secreted cytokine levels.

Functional profiling of CAR NK cells
Killing of specific tumor cell line by CAR NK cells in a ratio-dependent manner

Killing assay

To assess CAR NK cell–mediated cytotoxicity, CAR NK cells and a tumor cell line expressing the antigen of interest are co-cultured in several effector-to-target cell ratios. Tumor cells that do not express the antigen of interest are used as control. The killing rate is then determined by flow cytometric analysis of the viability of the specific tumor cell lines. 

Cytokine analysis of supernatant from CAR NK cells co-cultured with target cells using the MACSPlex Cytokine Kit

High-throughput and automated cytokine quantification for potency assessment

Accurate quantification of cytokines facilitates detailed potency examination of engineered CAR NK cells. MACSPlex Cytokine Kits were designed for easy quantification of secreted cytokines in cell culture supernatant, making it easy to assess CAR NK cell functionality in vitro. Up to 12 human cytokines can be analyzed in one sample using every standard multicolor flow cytometer. MACSPlex Cytokine Kits are furthermore optimized for automated measurement using the MACSQuant® Express Modes.

  • Bari, R. et al. (2019) A distinct subset of highly proliferative and lentiviral vector (LV)-transducible NK cells define a readily engineered subset for adoptive cellular therapy. Front. Immunol. 10: 2001.
  • Kloess S. et al. (2017) Optimization of human cell manufacturing: Fully automated separation, improved ex vivo expansion using IL-21 with autologous feeder cells, and generation of anti-CD123-CAR–expressing effector cells. Hum. Gene Ther. 28: 897–913
  • Kloess, S. et al. (2019) Preclinical assessment of suitable natural killer cell sources for chimeric antigen receptor natural killer–based “off-the-shelf” acute myeloid leukemia immunotherapies. Hum. Gene Ther. 30: 381–401.
  • Knorr, D.A. et al. (2013) Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy. Stem Cells Transl. Med. 2: 274–83.
  • Liu, E. et al. (2020) Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. N. Engl. J. Med 382: 545–53.
  • Nowakowska, P. et al. (2018) Clinical grade manufacturing of genetically modified, CAR-expressing NK-92 cells for the treatment of ErbB2-positive malignancies. Cancer Immunol. Immunother. 6725–38.  

Get acquainted with our protocol for engineering of CAR NK cells.


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