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. 2021 Sep;23(9):787-792.
doi: 10.1016/j.jcyt.2021.04.010. Epub 2021 Jun 9.

Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors

Peter Chockley  1 , Sagar L Patil  1 , Stephen Gottschalk  2
Affiliations

Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors

Peter Chockley et al. Cytotherapy. 2021 Sep.

Abstract

Background aims: Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors (LVs) are widely used to reliably generate genetically modified, clinical-grade T-cell products. However, the results of genetically modifying natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable transduction of NK cells by VSV-G LVs.

Methods: The authors activated NK cells from peripheral blood mononuclear cells using standard procedures and transduced them with VSV-G LVs encoding a marker gene (yellow fluorescent protein [YFP]) or functional genes (chimeric antigen receptors [CARs], co-stimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, amlexanox). NK cell transduction was evaluated by flow cytometry and/or western blot and the functionality of expressed CARs was evaluated in vitro.

Results: Blocking TBK1/IKKε during transduction of NK cells enabled their efficient transduction by VSV-G LVs as judged by YFPexpression of 40-50%, with half maximal effective concentrations of 1.1 µM (MRT67307), 5 µM (BX-795) and 24.8 µM (amlexanox). Focusing on MRT67307, the authors successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible co-stimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality.

Conclusions: The authors demonstrate that inhibition of TBK1/IKKε enables the reliable generation of genetically modified NK cells using VSV-G LVs. The authors' protocol can be readily adapted to generate clinical-grade NK cells and thus has the potential to facilitate the clinical evaluation of genetically modified NK cell-based therapeutics in the future.

Keywords: IKKε; TBK1; VSV-G; cancer; chimeric antigen receptor; immunotherapy; lentivirus; natural killer.

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Conflict of interest statement

Conflict of interest

SG has patent applications in the fields of T-cell and/or gene therapy for cancer. He has a research collaboration with TESSA Therapeutics, is a DSMB member of Immatics, and on the scientific advisory board of Tidal.

Figures

Figure 1:
Figure 1:. Viral pathway inhibition allows for VSV-G LV transduction of NK cells.
(A) Simplified endosomal TLR4 viral sensing pathway schematic in NK cells with indicated drug targeting. (B) Representative flow cytometry histogram plots of YFP expression in NK cells at highest used drugs concentrations (untreated: NK cells transduced in the absence of TBK1/IKKε inhibitors). (C) %YFP expression curves, N=3 mean±sem, in response to increasing drug concentrations; the half maximal effective concentration (EC50) for each compound is shown in parentheses.
Figure 2:
Figure 2:. CD19-CAR NK cells recognize and kill CD19-positive target cells.
(A) Scheme of CD19-CAR (GREEN: antigen recognition domain, GREY: hinge and transmembrane domain, ROSE: costimulatory domain, BLUE: signaling domain). (B) Representative flow cytometry histogram plot depiction CD19-CAR expression with indicated MOIs (untreated: NK cells transduced in the absence of MRT67307 at MOI of 30). (C) Flow cytometric based cytotoxicity assay of CD19-CAR NK cells targeting leukemia cell lines BV173 and BV173.CD19KO at indicated effector to target (E:T) ratios; N=4 donors, averaged technical triplicates per donor, mean±sem; unpaired two-tailed student’s t-test was used to determine significance, **p<0.01.
Figure 3:
Figure 3:. Viral pathway inhibition allows insertions of two transgenes into NK cells.
(A) Scheme of HER2-CAR.2A.iMC expression cassette (RED: antigen recognition domain, GREEN: hinge (H), GREY: transmembrane Domain (TM), BLUE: signaling domain, ORANGE: 2A sequence, GOLDENROD: iMC). (B) Representative flow cytometry plot demonstrating HER2-CAR expression detected with anti-F(ab’)2. (C) Western blot analysis of HER2-CAR NK cells demonstrating i) HER2-CAR.CD3δ and endogenous CD3δ (anti-CD3δ), and ii) iMC (anti-HA tag) expression. GAPDH was used a loading control. (D) 4-hour MTS cytotoxicity assay at an E:T ratio of 2:1, 1:1, and 0.5:1 with NK cells that were transduced with HER2-CAR LVs in the absence (untreated) or presence of MRT67307 (HER2-CAR) as effectors and A549 cells as targets, N=4 donors, averaged technical replicates per donor, mean±sem. Unpaired two-tailed student’s t-test was used to determine significance, *p<0.05. (E) Percentage of NK cells that produced 2, 3, 4, or 5+ effector molecules of a 32-analyte panel. Chemical Inducer of Dimerization: CID was used to initiate iMC signaling. (F) Polyfunctional Strength Index detailing effector, stimulatory, regulatory, chemo attractive, and inflammatory molecules; N=2 donors. (G) Aggregate data of NK cells transduced in the absence (−) (N=18) or presence (+) (BX-795 N=4, MRT67307 N=9) of TBK1/IKKε inhibitors; mean±sem; ****p<0.0001; Mann Whitney U-test.
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