‘Off the shelf’ immunotherapies: Generation and application of pluripotent stem cell‐derived immune cells

Abstract In recent years, great strides have been made toward the development of immune cell‐based therapies in the treatment of refractory malignancies. Primary T cells and NK cells armed with chimeric antigen receptors have achieved tremendous clinical success especially in patients with leukaemia and lymphoma. However, the autologous origin of these effector cells means that a single batch of laboriously engineered cells treats only a certain patient, leading to high cost, ununiform product quality, and risk of delay in treatment, and therefore results in restricted accessibility of these therapies to the overwhelming majority of the patients. Addressing these tricky obstacles calls for the development of universal immune cell products that can be provided ‘off the shelf’ in a large amount. Pluripotent stem cells (PSCs), owing to their unique capacity of self‐renewal and the potential of multi‐lineage differentiation, offer an unlimited cell source to generate uniform and scalable engineered immune cells. This review discusses the major advances in the development of PSC‐derived immune cell differentiation approaches and their therapeutic potential in treating both hematologic malignancies and solid tumours. We also consider the potency of PSC‐derived immune cells as an alternative therapeutic strategy for other diseases, such as autoimmune diseases, fibrosis, infections, et al.

autologous origin of these effector cells means that a single batch of laboriously engineered cells treats only a certain patient, leading to high cost, ununiform product quality, and risk of delay in treatment, and therefore results in restricted accessibility of these therapies to the overwhelming majority of the patients. Addressing these tricky obstacles calls for the development of universal immune cell products that can be provided 'off the shelf' in a large amount. Pluripotent stem cells (PSCs), owing to their unique capacity of self-renewal and the potential of multilineage differentiation, offer an unlimited cell source to generate uniform and scalable engineered immune cells. This review discusses the major advances in the development of PSC-derived immune cell differentiation approaches and their therapeutic potential in treating both hematologic malignancies and solid tumours. We also consider the potency of PSC-derived immune cells as an alternative therapeutic strategy for other diseases, such as autoimmune diseases, fibrosis, infections, et al.

| INTRODUCTION
As a targeted therapeutic approach, immune cell therapy is in rapid development and has become a promising treatment modality with the potential to cure a wide range of diseases. [1][2][3] At present, nearly a dozen of immune cell therapies have been approved worldwide, most of which are for cancer patients. Notably, all these approved drugs are autologous products, derived from the patient's own peripheral blood. Although peripheral blood cells are readily available and the immune rejection responses of these engineered cells are minimal, the disadvantages are obvious: engineering of patient-derived immune cells results in high heterogeneity, high cost, and long manufacturing time, which ultimately leads to restricted patient accessibility. To this end, several groups are developing allogeneic immune cell products derived from peripheral blood of healthy donors. In this way, a single batch of drugs can be distributed to a number of patients. However, the expansion capacity of these allogeneic immune cells is still limited.
Moreover, to avoid host-versus-graft (HvG) reaction and graft-versus-host disease (GvHD), primary immune cells generally need to be genetically modified to knock out HLA molecules and, in the case of T cells, to disrupt TCR. In addition, in the treatment of diseases such as cancer, genetic modification for integration of the CAR transgene is often required to improve the function of Chenxin Wang and Jingjing Liu contributed equally to this study. immune cells. 4 These genetic modifications in primary cells might increase the risk of tumorigenesis because of a lack of safety certification after genome editing and moreover, might reduce the yield of the final product because of the limited editing efficiencies.
Pluripotent stem cells (PSCs), whether embryonic stem cells (ESCs) 5,6 or induced pluripotent stem cells (iPSCs), 7 have unlimited self-renewal capacity and can be differentiated into various types of immune cells, thus provide an ideal cell source for developing 'off-the-shelf' cell therapies. Moreover, PSCs provide a convenient platform for genetic modifications at the starting stage, as properly edited cells can be isolated and identified from individual clones and evaluated for off-target genomic alterations through whole-genome sequencing. Once certified, a single genetically modified PSC clone can be expanded and differentiated into engineered functional immune cells at a nearly unlimited scale. 8,9 With the combination of the PSC techniques, efficient lineage-specific immune cell differentiation approaches, and safe and efficient gene editing methods, PSCs offer an alternative accessible platform to produce engineered immune cells in large amount (Figure 1). At present, approaches for the manufacturing of multiple PSC-derived immune cells that can be scaled up without serum or feeder cells have also been established. A variety of PSC-derived immunotherapeutic products have also entered clinical trials. 10 In this review, we summarize the advances in immune cell differentiation methods (Table 1) and clinical applications of these engineered immune cells derived from human PSCs (Table 2).

| PSC-DERIVED T CELLS
Although the application of engineered primary T cells has achieved great success in the clinic, PSC-derived T cells are faced with additional challenges because T cell development involves TCR rearrangement and positive/negative selection in vivo. Generation of PSC-derived T cells involves a complicated process and requires two essential stages: PSCs are differentiated into haematopoietic progenitor cells or haematopoietic stem cells (HPCs/HSCs) in the first stage and then, HPCs/HSCs are committed into T cell lineages via Notch signalling in the second stage. 37 Galic demonstrated that human ESCs can be specified into a T lymphoid lineage by coculture with feeder cells and subsequent engraftment into thymic tissues in immunodeficient mice. 38,39 However, these T cells were limited in cell number and their functions were not evaluated. Mouse iPSC-derived functional T cells were first generated by Lei et al. via coculture with stromal cells OP9-DL1. 40 These iPSC-derived T cells secreted IL-2 and IFNγ after activation and restored the T-cell pool in Rag1-deficient mice. In 2014, Kishino generated transgene-free human peripheral blood T cell-derived iPSCs in a defined culture medium and a feeder-free condition, making them more suitable for therapeutic applications. 41 In a more recent study, iPSC-derived T cells were demonstrated to be capable of expanding up to 10,000-fold. Furthermore, these T cells expressed CD8αβ + and exhibited improved antigenspecific cytotoxicity compared with CD8αα + T cells and prolonged the survival of murine models with tumours. 42 iPSCs derived from non-T cells (non-T iPSCs), such as fibroblasts and primary skin cells, bear unrearranged TCR. After T cell F I G U R E 1 Schematic model of strategies and platforms for generation of peripheral blood-derived and PSC-derived immune cells. Based on the unique advantages of PSCs, multiple genetic modifications can be introduced to produce safe, versatile, abundant and functionally enhanced immune cells for clinical applications.
T A B L E 1 Representative differentiation methods for generating PSC-derived immune cells. In vitro phagocytosis and cytokine production assays 36 followed by generation of CD8αβ T cells with antigen-specific cytotoxic activity comparable to CD8αβ T regenerated from T-iPSCs, similarly inhibited tumour growth in xenograft tumour models. 46 In 2013, antigen-specific T cells derived from iPSCs were generated. 12,13,47 Nishimura T amplifies antigen-specific CD8 T cells from HIV-infected patients and then sub-induces them into iPSCs (T-iPSCs). Further redifferentiation generated CD8 T cells with high proliferative capacity, elongated telomeres, and antigen-specific killing activity. 12  Furthermore, the 3D-organoid systems, such as artificial thymic organoid (ATO) and fetal thymic organ culture systems, were established for the generation of mature T cells derived from PSCs. 16,51,[53][54][55][56] In particular, the ATO-based differentiation system was used to induce T-iPSCs derived from CD62L + naive and memory T cells into CD8αβ + T cells. These T cells showed comparable antigenspecific activation, degranulation, cytotoxicity, and cytokine secretion capabilities to conventional engineered primary T cells. 51 Other strategies have enhanced T cell-specific lineage commitment via genetic modifications in iPSCs. EZH1 knockdown facilitates differentiation and maturation of T cells from iPSCs in vitro, displaying a highly diverse TCR repertoire and mature molecular signatures similar to TCRαβ T cells from peripheral blood. 57 133 and inducible T cell co-stimulator (ICOS). 134 The fourth generation of CARs, also referred to as armoured CARs, produces an additional protein molecule, such as cytokines for tuning of CAR signalling to enhance proliferation and persistence of CAR T cells in the body. 135,136 To date, the Clinical trials of iPSC-derived CAR T cells are in progress and the Fate Therapeutics company is a pioneer in this field. Fate Therapeutics have developed a feeder-free differentiation protocol to generate scalable iPSC-derived CAR T cells. In the FT819 CAR T product, a CD19 CAR was integrated into the TCRα locus to obtain regulated CAR expression and to overcome GvHD effect. The CD3ζ module of the CAR construct was also modified to achieve prolonged persistence of the engineered CAR T cells. 126  Upon forming immunological connections with a target, the behaviour of an NK cell is determined by the cumulative signal generated from engaged receptors toward ligands on the target cell. 146 Once the target cell recognition is complete, NK cells elicit a 'killing' response through the release of cytolytic granules and cytotoxic cytokines. 147 Moreover, they may also conduct antibody-dependent cellular cytotoxicity (ADCC) when their CD16 receptor is engaged with antibody-coated cells. 148 NK cells play a pivotal role in anti-cancer immunity and have been used as immunotherapeutic agents in cancer therapies. Given the limited numbers of primary NK cells that can be purified from autologous sources, differentiation of NK cells from PSCs provides approaches for large-scale production of these cells for immuno-therapeutic applications. While generation of functional PSC-derived T cells is indeed inefficient, protocols for NK cell production from PSCs are now much more routinely developed. 19,73,75,149 Unlike the therapeutic T cell products that recognized non-self-host   58 The iPSC-derived Treg cells were also retrovirally transduced with autoantigen-specific TCRs to generate Ag-specific iPSC-Tregs, which exhibited abilities to suppress the development of autoimmune arthritis and T1D after adoptive transfer in murine models. 59,169 Targeting Ag-presenting cells, especially the self-killing infiltrating T cells with engineered CAR T cells is also expected to have therapeutic potential in autoimmune diseases.
In addition to application in cancer therapies, iPSC-derived macrophages provide a promising avenue for ameliorating fibrosis in degenerative organs and elimination of microbial infections. Somayeh Pouyanfard et al. differentiated human iPSCs into macrophages that exhibit classical surface cell markers and phagocytic activity similar to their peripheral blood-derived counterparts. Moreover, they demonstrated that these cells were efficiently polarized to pro-inflammatory M1 or anti-inflammatory M2 phenotypes in presence of LPS + IFN-γ and IL-4 + IL-13, respectively. They also observed that the M2 iPSCderived microphages significantly reduce fibrogenic gene expression and disease-associated histological markers including Sirius Red, αSMA, and desmin in immunodeficient mice models. 170 Staphylococcus aureus is a common offending organism that causes respiratory infections in the lung. Recent studies have exploited iPSC-derived macrophages in the treatment of pulmonary S. aureus infections.
Adoptive transfer of iPSC-derived macrophages resulted in reduced bacterial load, reduced granulocyte infiltration, and less damage in lung tissue in S. aureus-infected immunodeficient murine models. 25,171 In addition, PSC-derived DCs can also participate in the regulation of

AUTHOR CONTRIBUTION
CXW and JJL involved in literature collection and manuscript preparation. WL supervised this work.