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

Abstract

Background Redirection 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. Methods In 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. Results We 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. Conclusion Looking 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.

Advances in NK cell therapy for brain tumors

“…This process is lengthy and expensive and leaves the possibility of adverse events including cytokine release syndrome (CRS). Alternatively, NK CAR products can be derived from haploidentical, patient-derived, ex vivo expanded NK 90 , NK cell lines such as NK92 91 – 93 or from iPSC-derived NK cell products 94 , 95 constituting an off-the-shelf product that can be administered regardless of patient MHC background and have demonstrated limited adverse effects compared to CAR-T therapies 96 . Thus, NK cells represent a robust and flexible platform for a variety of CARs that may target CNS tumor-specific antigens, with less neurotoxicity and cytokine release syndrome seen with CAR-T cell products.…”

Adoptive Immunotherapy: A Human Pluripotent Stem Cell Perspective

“…FT596, the first iPSC-derived CAR NK cell product candidate that is currently under clinical trial, utilized the same CAR construct to target CD19 + Bcell lymphoma [Goodridge et al, 2019]. On the other hand, CD28 and 4-1BB costimulatory domains and CD3ζ intracellular domain were used to generate anti-EpCAM and anti-GPC3 CAR NK cells derived from human iPSCs [Ueda et al, 2020;Tang et al, 2021]. CD28 and 4-1BB domains, typically utilized for T cells, were still effective in mediating cytotoxicity of NK cells against EpCAM + and GPC3 + cancer cells.…”

“…The transmembrane domain of NKG2D, a NK cell-activating receptor, costimulatory domain of 2B4, a mediator of cytotoxicity in NK cells, and a CD3ζ signaling domain improved the efficacy of CAR NK cells in tumor killing. Anti-GPC3 [Ueda et al, 2020] and anti-EpCAM [Tang et al, 2021] CAR NK cells derived from human iPSCs also exhibited specific cytotoxicity against targeted tumor cells. More than 10 clinical trials are ongoing for hPSC-derived NK cells with or without CAR engineering (Table 2).…”

Understanding the versatile roles and applications of EpCAM in cancers: from bench to bedside

“…Cotreated with regorafenib improves EpCAM CAR NK-92 cells by enhancing immune cell infiltration, downregulating immune suppressive cell subsets and improving tumor vasculature [ 152 ]. Additionally, induced EpCAM CAR NK cells derived from modified induced pluripotent stem cells displays anti-tumor activities as well, which provides a novel approach to generate CAR NK cells more efficiently [ 153 ].…”