SummaryCurrent donor cell-dependent strategies can only produce limited “made-to-order” therapeutic natural killer (NK) cells for limited patients. To provide unlimited “off-the-shelf” NK cells that serve many recipients, we designed and demonstrated a holistic manufacturing scheme to mass-produce NK cells from induced pluripotent stem cells (iPSCs). Starting with a highly accessible human cell source, peripheral blood cells (PBCs), we derived a good manufacturing practice-compatible iPSC source, PBC-derived iPSCs (PBC-iPSCs) for this purpose. Through our original protocol that excludes CD34+ cell enrichment and spin embryoid body formation, high-purity functional and expandable NK cells were generated from PBC-iPSCs. Above all, most of these NK cells expressed no killer cell immunoglobulin-like receptors (KIRs), which renders them unrestricted by recipients' human leukocyte antigen genotypes. Hence, we have established a practical “from blood cell to stem cells and back with less (less KIRs)” strategy to generate abundant “universal” NK cells from PBC-iPSCs for a wide range of patients.
Using induced pluripotent stem cells (iPSCs) to derive chimeric antigen receptor-modified T (CAR-T) cells has great industrial potential. A previous study used αβ T cell-derived CAR-modified iPSCs to produce CAR-T cells. However, these αβ T cells are restricted to autologous use and only recognize single cancer antigen. To make CAR-T alternative for allogeneic use, we reprogrammed γδ T cell into iPSCs (γδ T-iPSCs) to circumvent the risk of graft-versus-host disease. To target multiple cancer-associated antigens, we used an “NK cell-promoting” protocol to differentiate γδ T-iPSCs and to induce expression of natural killer receptors (NKRs). Through such two-step strategy, mimetic γδ T cells endowed with an array of NKRs and thus designated as “γδ natural killer T (γδ NKT) cells” were derived. With no/low-level expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) and immune checkpoint receptors, γδ NKT cells may provide a potent “off-the-shelf” cytotoxic cell source to recognize multiple ubiquitous antigens in a broad spectrum of cancers.
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.
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