Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells

Shin Yi Tang,Yumei Luo,Zhicheng Du,Jieming Zeng,Shijun Zha,Detu Zhu,Shu Wang

doi:10.1186/s13287-021-02648-4

Abstract

BackgroundRedirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment. However, the site-specific integration of a CAR gene into NK cells remains challenging.MethodsIn the present study, we genetically modified human induced pluripotent stem cells (iPSCs) with a zinc finger nuclease (ZFN) technology to introduce a cDNA encoding an anti-EpCAM CAR into the adeno-associated virus integration site 1, a “safe harbour” for transgene insertion into human genome, and next differentiated the modified iPSCs into CAR-expressing iNK cells.ResultsWe detected the targeted integration in 4 out of 5 selected iPSC clones, 3 of which were biallelically modified. Southern blotting analysis revealed no random integration events. iNK cells were successfully derived from the modified iPSCs with a 47-day protocol, which were morphologically similar to peripheral blood NK cells, displayed NK phenotype (CD56+CD3-), and expressed NK receptors. The CAR expression of the iPSC-derived NK cells was confirmed with RT-PCR and flow cytometry analysis. In vitro cytotoxicity assay further confirmed their lytic activity against NK cell-resistant, EpCAM-positive cancer cells, but not to EpCAM-positive normal cells, demonstrating the retained tolerability of the CAR-iNK cells towards normal cells.ConclusionLooking ahead, the modified iPSCs generated in the current study hold a great potential as a practically unlimited source to generate anti-EpCAM CAR iNK cells.

Highlights

Redirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment
To construct the donor vector for Adenoassociated virus integration site 1 (AAVS1) site homologous recombination, the complete sequence of a cytomegalovirus (CMV) promoter driving the expression of anti-Epithelial cell adhesion molecule (EpCAM) CAR from the mRNA CAR vector [24] was subcloned into pFB-EF1αEGFP-AAVS1 donor vector developed previously [18] containing a eukaryotic translation elongation factor 1 alpha (EF1α) promoter driving Green fluorescence protein (GFP) expression cassette and a mouse phosphoglycerate kinase 1 (PGK) promoter driving the neomycin resistant gene expression cassette, which was flanked by homologous sequence of AAVS1 locus (Fig. 1A)
We designed a donor sequence encoding a cytomegalovirus (CMV) promoter driven thirdgeneration anti-EpCAM CAR, which was comprised of a humanized single-chain variable fragment 4D5MOC-B [27], a CD8 alpha hinge transmembrane region, two co-stimulatory domains (CD28 and 4-1BB) and a CD3 zeta T cell activation domain, as shown in Additional file 1: Fig. S1

Summary

Introduction

Redirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment. Unlike the success and feasibility in CAR-T cell generation, CAR-NK cell generation from primary NK cells is faced with key obstacles such as the cost- and time-consuming expansion of primary NK cells and the relatively low efficiency of genetic modification [5,6,7,8,9]. To address these challenges, induced pluripotent stem cells (iPSCs) have been tested as an unlimited cell source for NK cell generation [10,11,12,13]. We examined whether the CAR gene-modified iPSCs were capable of differentiating into CAR-expressing NK cells

Methods

Results

Discussion

Conclusion