Design and analysis of stably integrated reporters for inducible transgene expression in human T cells and CAR NK-cell lines

Abstract

BackgroundCytotoxic activity of T- and NK-cells can be efficiently retargeted against cancer cells using chimeric antigen receptors (CARs) and rTCRs. In the context of solid cancers, use of armored CAR T- and NK cells secreting additional anti-cancer molecules such as cytokines, chemokines, antibodies, BiTEs, inverted cytokine receptors, and checkpoint inhibitors, appears particularly promising, as this may help overcome immunosuppressive tumor microenvironment, attract bystander immune cells, and boost CAR T/NK-cell persistence. Placing the expression of such molecules under the transcriptional control downstream of CAR-mediated T/NK-cell activation offers the advantage of targeted delivery, high local concentration, and reduced toxicity. Several canonic DNA sequences that are known to function as activation-inducible promoters in human T and B cells have been described to date and typically encompass the multimers of NFkB and NFAT binding sites. However, relatively little is known about the DNA sequences that may function as activation-driven switches in the context of NK cells. We set out to compare the functionality of several activation-inducible promoters in primary human T cells, as well as in NK cell lines NK-92 and YT.MethodsLentiviral constructs were engineered to express two fluorescent reporters: mCherry under 4xNFAT, 2xNFkB, 5xNFkB, 10xNFkB, 30xNFkB promoters, as well as two variants of the CD69 promoter, and copGFP under the strong constitutive promoter of the human EF1a gene. Pseudotyped lentiviral particles obtained using these constructs were transduced into primary human T cells and NK-92 and YT cell lines expressing a CAR specific for PSMA. The transgenic cells obtained were activated by CD3/CD28 beads (T cells) or via a CAR (CAR-NK cell lines). Promoter activity before and after activation was assayed using FACS analysis.ResultsIn T cells, the CD69 promoter encompassing CNS1 and CNS2 regions displayed the highest signal/noise ratio. Intriguingly, in the context of CAR-YT cell line neither of the seven promoters tested displayed acceptable activation profile. In CAR-NK-92 cells, the largest fold activation (which was modest) was achieved with the 10xNFkB and 30xNFkB promoters, however its expression was clearly leaky in “resting” non-activated cells.ConclusionsUnlike in T cells, the robust activation-driven inducible expression of genetic cassettes in NK cells requires unbiased genome-wide identification of promoter sequences.Electronic supplementary materialThe online version of this article (10.1186/s12920-019-0489-4) contains supplementary material, which is available to authorized users.

Design and Characterization of an “All-in-One” Lentiviral Vector System Combining Constitutive Anti-GD2 CAR Expression and Inducible Cytokines

“…Stably integrated reporter cell lines for transgene expression harboring activation-inducible promoters were already described for functional testing of T or natural killer (NK) cell activation, as well as in the context of CAR T and NK cell therapy [42,43]. These reporters are mostly based on NFAT-, nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB)-, and activator protein (AP)-1-based promoters, as activation pathways in T and NK cells are mediated by these transcription factors.…”

“…Another potential application for the "all-in-one" constructs is to screen CARs and TCRs in reporter assays [42][43][44] by displaying CAR libraries, including different scFvs, hinge regions, and signaling domains directly in primary human immune cells. The CAR function can be directly linked to the NFAT promoter-driven iGOI expression or cytokine secretion and can be investigated after target antigen recognition.…”

Toward Tightly Tuned Gene Expression Following Lentiviral Vector Transduction

“…Although externally-provided cues can be used to control transgene expression through specific promoters, the development of completely autonomous inducible systems is the focus of intense research efforts currently. In this direction, it should be noted that CAR T cells efficacy can be potentiated by combining the direct attack of the CAR T cells themselves along with the secretion of ectopic cytokines in order to boost the immune response against target components (CAR T fourth generation, TRUCK CAR) [ 122 , 123 ]. This is achieved by inserting cytokine transgenes under the control of a Nuclear Factor of Activated T-cells (NFAT)-inducible promoter, since CAR stimulation induces, phosphorylation of NFAT, which subsequently migrates to the nucleus and activates sensitive promoters inserted in the LV construct and hence, gene expression.…”

Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy

“…Currently, there is only one comparison of different promotors on CAR expression and function in NK cell lines published, while no comparative data are available for primary NK cells [ 33 ]. From the single report on CAR-NK cells, no definitive statements can be made about the best promotor for CAR-NK cells [ 33 ]. In primary T cells, various comparisons of the effect of promoters on CAR expression levels were performed.…”