Single-Cell Functional Proteomic Characterization of CAR NK Cells Sheds Light on Improved Transduction Method

The development of CAR-NK cells is sparking a great deal of interest among researchers – primarily due to NK cells possessing unique advantages over traditional CAR-T cells. Unlike CAR-T cells, NK cells can be applied in an allogeneic cell therapy setting, which means it has the potential of becoming an “off-the-shelf” therapy as well as reducing the risk of graft versus host disease (GVHD). CAR-NK cells don’t persist in vivo as long as CAR-T cells do, thus reducing the risk of on-target/off-tumor toxicity. In addition, the risk of cytokine release syndrome (CRS) and neurotoxicity is lower because of the difference in cytokines secreted from NK cells as compared to T cells.1

What’s more, the expression of a CAR in NK cells can increase their inherent cytotoxicity and improve on disappointing results that can often be obtained with unmodified NK cells. While preclinical studies with unmodified NK cells have yielded promising results, they have been difficult to translate into successful clinical trials. Clinical trials using unmodified NK cells have confirmed their safety for patients. However, while some clinical trials with unmodified NK cells have reported clinical success, producing positive responses in up to 50% of patients, other trials have reported limited clinical efficacy, suggesting that the use of a CAR could dramatically enhance NK cell therapies.2

NK Cell Transduction with Lentivirus

One major challenge limiting the development of CAR-NK cell therapies is efficient gene transfer. Retroviral vectors have been widely studied and typically have an efficiency of approximately 27–52% in NK cells after one round of transduction. However, the use of retroviral vectors in clinical development of CAR-NK cells is limited due to issues with low cell viability after transduction and the risk of insertional mutagenesis.

A viable, safer option is transduction with lentivirus (LV) as it provides a reduced risk of genotoxicity and insertional mutagenesis. However, the transduction efficiency is often much lower as compared to retroviral transduction.

There are several types of LV under development for CAR-NK cells, with recent interest in optimizing transduction with vesicular stomatitis virus-G (VSV-G)-pseudo typed LV (VSV-G LVs).

New studies suggest that the poor transduction efficiency and low cell viability in NK cells transduced with VSV-G LVs is due to activation of the Toll-like receptor 4 (TLR4) pathway, a finding that the authors of a recent study wanted to explore further.3

VSV-G Activation of the TLR4 Signaling Pathway

The immune system uses pattern recognition receptors, such as toll-like receptors, to recognize the pathogen-associated molecular patterns (PAMPs) of bacteria and viruses. TLR4 is known for its ability to recognize lipopolysaccharide (LPS) on the outer membrane of gram-negative bacteria, however it can recognize viruses as well.

NK cells express TLR4 both on the cell surface and intracellularly,4 making these cells especially sensitive to the activation of the TLR4 pathway by VSV-G LV. VSV-G activates the endosomal TLR4 signaling pathway, which induces a signaling cascade that ultimately results in the production of type I interferons. Based on previous studies, it was thought that poor transduction efficiency of VSV-G LV could be due to activation of the TLR4 pathway. Thus, the Chockley et al study investigated the use of the drug MRT67307 to inhibit TBK1 and IKKε, two molecules that signal downstream in the TLR4 pathway.

Single-Cell Functional Proteomics in the Evaluation of Gene-Edited NK Cells

Chockley et al showed that the use of MRT67307 in the transduction protocol resulted in significantly improved transduction efficiencies of a HER2-CAR in NK cells, with up to 53% of cells expressing the CAR.

Additionally, cytotoxic capacity was significantly improved for HER2-CAR NK cells transduced in the presence of MRT67307.

IsoPlexis’ Single-Cell Secretome Solution was used to assess cytokine secretion profiles of HER2-CAR NK cells, in comparison to unmodified NK cells. CAR NK cells secreted a range of effector cytokines including: granzyme B, perforin, TNF-α, MIP1-β, MCP-1, RANTES, and IL-8. In contrast, less than 13% of unmodified cells secreted granzyme B and less than 3.5% secreted the other cytokines. Additionally, unmodified NK cells secreted 2–3 effector molecules on average, while CAR NK cells were capable of producing up to 10 effector molecules.

Data from this study suggests that inhibition of TBK1 and IKKε significantly improves the transduction of NK cells and results in a fully functional cell product, with a superior cytotoxic capacity in comparison to both unmodified NK cells and NK cells transduced in the absence of MRT67307.

Utilizing data uncovered with Isoplexis’s single cell proteomics platform, the authors were able to characterize reliable generation of genetically modified NK cells using VSV-G LVs.3 The study’s NK cell transduction method could potentially be adapted to clinical production of allogeneic CAR NK cell therapies, which has large implications in the field of off-the-shelf cell therapy research.



  1. Xiea, G., et al. CAR-NK cells: A promising cellular immunotherapy for cancer. EBioMedicine. 59 (2020) 102975.
  2. Shimasaki N, Jain A, Campana D. NK cells for cancer immunotherapy. Nat Rev Drug Discov. 2020 Mar;19(3):200-218. doi: 10.1038/s41573-019-0052-1. Epub 2020 Jan 6. PMID: 31907401.
  3. Chockley P, Patil SL, Gottschalk S. Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors. Cytotherapy. 2021 Jun 9:S1465-3249(21)00645-9. doi: 10.1016/j.jcyt.2021.04.010. Epub ahead of print. PMID: 34119434.
  4. Adib-Conquy, M. et al. TLR-mediated activation of NK cells and their role in bacterial/viral immune responses in mammals. 2014. 92:3(256-262).
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