Differentiation of human induced pluripotent stem cells expressing chimeric antigen receptors into distinct immune cells as an off-the-shelf strategy for tumor treatments

Abstract Immunotherapy based on T cells expressing Chimeric Antigen Receptor (CAR- T) has been shown to be a promising strategy for treating hematological malignancies. However, its efficacy in solid tumors remains limited. Additionally, the personalized manufacturing of autologous CAR-T cells presents logistical and biological challenges, prompting the need for universal, “off-the-shelf” alternatives. In this context, induced pluripotent stem cells (iPSCs) represent a promising platform to generate different CAR-engineered immune effectors from a single, genetically defined source. In this study, we present the use of CAR+ iPSC as a multi-functional platform for the generation of diverse immune cells with complementary antitumor activities, including T lymphocytes, natural killer (NK) cells, and dendritic cells (DCs). iPSCs were genetically modified via electroporation using the non-viral piggyBac transposon system. Although we obtained only 2% of CAR efficiency, after two rounds of CAR+ iPSC sorting followed by clone selection, we achieved a purity of 95%, with stable CAR expression over 40 days. CAR+ iPSCs were differentiated to hematopoietic progenitor-like cells by embryoid body (EB) formation in ultra- low attachment conditions. Hematopoietic progenitors obtained at appropriate time points were then used for lineage-specific differentiation into T, NK, and DC cells under defined cytokine environments. To explore the full potential of this CAR+ iPSC platform, we established lineage- specific differentiation protocols using wild type iPSCs to generate dendritic cells, T lymphocytes, and NK cells, each with complementary immunological functions. For DC differentiation, hematopoietic cells were cultured with GM-CSF and IL-4, leading to typical myeloid immune cell morphology, including a few macrophage-like cells, with large cytoplasmatic vacuoles, and numerous cells with dendrite projections close to DC morphology and expression of HLA- DR+CD64LowXCR1High, resembling cDC1-like cells. Preliminary data shows that these cDC1-like cells can induce CD3+ T cells proliferation in vitro in a similar way to the control using moDC. Differentiation of CAR⁺ iPSCs into DCs is ongoing, and CAR expressions will be tracked throughout myeloid development to assess their antigen-presenting and antitumor potential. Simultaneously, T cell differentiation was achieved by culturing hematopoietic progenitors under feeder- free conditions or co-culturing them with monocyte-derived DCs (Mo-DCs), in the presence or absence of CD3/CD28 activation. As expected, the standard protocol predominantly yielded CD8αα+TCRγδ+CD7+CD5- T cells. In contrast, Mo-DC co-culture with CD3/CD28 stimulation enabled the generation and expansion of CD4+ T cells expressing either TCRαβ or TCRγδ, with phenotypes including CD5+CD7- and CD5+CD7+. Upon stimulation, both CD4+ and CD8+ T cells produced IFN-γ, TNF-α, and TGF-β1, individually or in combination. This strategy increased the frequency of CD4⁺ T cells by the end of differentiation, although further analyses are needed to clarify their developmental origin. Finally, NK cells were generated by EB-derived hematopoietic cells in NK differentiation medium for four weeks. Flow cytometry confirmed the generation of CD56+CD16+CD3- NK cells expressing CD107a, indicative of cytotoxic function. Notably, genetic identity was retained throughout NK differentiation, as demonstrated by short tandem repeat (STR) profiling, supporting the use of iPSCs as a stable source for CAR+ NK cell generation. Differentiation of CAR+ iPSCs into NK cells is on going and we could observe that the CAR expression is maintained on hematopoietic progenitor cells generated. Together, these results demonstrate that CAR+ iPSCs represent a robust platform for the scalable generation of diverse immune effector cells with antitumor potential. The ability to produce CAR-T, CAR-NK, and CAR-DCs from the same parental iPSC clone supports the development of distinct immune strategies to target tumors through complementary mechanisms, enhancing immune activation and addressing resistance, particularly in solid tumors.