Differentiation of BCMA-specific induced pluripotent stem cells into rejuvenated CD8αß+ T cells targeting multiple myeloma.

ABSTRACT: A major hurdle in adoptive T-cell therapy is cell exhaustion and failure to maintain antitumor responses. Here, we introduce an induced pluripotent stem cell (iPSC) strategy for reprogramming and revitalizing precursor exhausted B-cell maturation antigen (BCMA)-specific T cells to effectively target multiple myeloma (MM). Heteroclitic BCMA72-80 (YLMFLLRKI)-specific CD8+ memory cytotoxic T lymphocytes (CTL) were epigenetically reprogrammed to a pluripotent state, developed into hematopoietic progenitor cells (CD34+ CD43+/CD14- CD235a-), differentiated into the T-cell lineage and evaluated for their polyfunctional activities against MM. The final T-cell products demonstrated (1) mature CD8αß+ memory phenotype, (2) high expression of activation or costimulatory molecules (CD38, CD28, and 41BB), (3) no expression of immune checkpoint and senescence markers (CTLA4, PD1, LAG3, and TIM3; CD57), and (4) robust proliferation and polyfunctional immune responses to MM. The BCMA-specific iPSC-T cells possessed a single T-cell receptor clonotype with cognate BCMA peptide recognition and specificity for targeting MM. RNA sequencing analyses revealed distinct genome-wide shifts and a distinctive transcriptional profile in selected iPSC clones, which can develop CD8αß+ memory T cells. This includes a repertoire of gene regulators promoting T-cell lineage development, memory CTL activation, and immune response regulation (LCK, IL7R, 4-1BB, TRAIL, GZMB, FOXF1, and ITGA1). This study highlights the potential application of iPSC technology to an adaptive T-cell therapy protocol and identifies specific transcriptional patterns that could serve as a biomarker for selection of suitable iPSC clones for the successful development of antigen-specific CD8αß+ memory T cells to improve the outcome in patients with MM.

Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells.

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such "hu-NK mice" rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

Improved anti-solid tumor response by humanized anti-podoplanin chimeric antigen receptor transduced human cytotoxic T cells in an animal model.

Recently, research has been conducted with chimeric antigen receptor (CAR)-T cells to improve efficacy against solid tumors. Humanized CAR improved the long-term survival of CAR-T cells in patients' peripheral blood, resulting in increased therapeutic efficacy. Therefore, the humanization of the CAR-gene sequence is considered an effective method. Podoplanin (PDPN) is a glycosylated transmembrane protein that is highly expressed in solid tumors and is associated with poor prognosis in patients with cancer. Therefore, PDPN is considered a biomarker and good target for cancer treatment with CAR-T cells. Previously, an anti-PDPN CAR was generated from a conventional nonhumanized antibody-NZ-1, the only anti-PDPN antibody for which a CAR was produced. In this study, we investigated other anti-PDPN CARs from the antibody NZ-27, or humanized NZ-1, to enhance the therapeutic potential of CAR-T cells. The CAR signal intensity was enhanced by the efficient expression of CAR proteins on the T-cell surface of NZ-27 CAR-T cells, which show tumor-specific cytotoxicity, proinflammatory cytokine production, and anti-tumor activity against PDPN-expressing tumor xenografts in mice that were significantly better than those in nonhumanized NZ-1 CAR-T cells.

Sustainable Antiviral Efficacy of Rejuvenated HIV-Specific Cytotoxic T Lymphocytes Generated from Induced Pluripotent Stem Cells.

Persistence of HIV latently infected cells is a barrier to HIV cure. The "kick and kill" strategy for a cure includes clearance of the viral reservoir by HIV-specific cytotoxic T lymphocytes (CTLs). However, exhaustion and senescence of T cells accelerates during HIV infection, and does not fully recover, despite complete viral suppression under antiretroviral therapy. We previously established an induced pluripotent stem cell (iPSC) from a parental HIV-specific CTL clone and generated an iPSC-derived rejuvenated HIV-specific CTL clone (iPSC-CTL), which exhibited an early memory phenotype, high proliferation capacity and effector functions in vitro. Here, we assessed the antiviral efficacy of the HIV-specific iPSC-CTL by single- and multiple-round viral suppression assays (VSAs). The HIV-specific iPSC-CTL suppressed viral replication in an HLA-dependent manner with equivalent efficacy to the parental CTL clone in single-round VSA. In multiple-round VSA, however, the ability of the iPSC-CTL to suppress viral replication was longer than that of the parental CTL clone. These results indicate that HIV-specific iPSC-CTL can sustainably exert suppressive pressure on viral replication, suggesting a novel approach to facilitate clearance of the HIV reservoir via adoptive transfer of rejuvenated CTLs. IMPORTANCE Elimination of latently HIV-infected cells is required for HIV cure. In the "kick and kill" strategy proposed for a cure to HIV, the host immune system, including HIV-specific cytotoxic T lymphocytes (CTLs), play a central role in eliminating HIV antigen-expressing cells following reactivation by latency-reversing agents (LRAs). However, CTL dysfunction due to exhaustion and senescence in chronic HIV infection can be an obstacle to this strategy. Adoptive transfer with effective HIV-specific CTLs may be a solution of this problem. We previously generated an induced pluripotent stem cell (iPSC)-derived rejuvenated HIV-specific CTL clone (iPSC-CTL) with high functional and proliferative capacity. The present study demonstrates that iPSC-CTL can survive and suppress HIV replication in vitro longer than the parental CTL clone, indicating the potential of iPSC-CTL to sustainably exert suppressive pressure on viral replication. Adoptive transfer with rejuvenated HIV-specific CTLs in combination with LRAs may be a new intervention strategy for HIV cure/remission.

[Basic Research and Clinical Development of Regenerative Immunotherapy Using iPS Cells].

In recent years, cell-based immunotherapies, such as chimeric antigen receptor(CAR)-T cell therapy, have greatly advanced the treatment of some hematological malignancies, especially those resistant to other therapies. Nevertheless, there are significant obstacles to the clinical application of current autologous therapies, such as high cost, challenging large-scale manufacturing, and difficulty obtaining long-term therapeutic efficacy due to T cell exhaustion. Induced pluripotent stem(iPS)cells have the potential to solve these problems through their unlimited proliferative capacity and differentiation potency to every type of cell in a body. Furthermore, iPS cells can be genetically engineered and differentiated into various types of immune cells, providing an unlimited resource for the development of"off-the-shelf"cell therapies. Here, we review the clinical development status of regenerative immunotherapies using iPS cell-derived CD8 killer T cells and natural killer(NK)cells and outline regenerative immunotherapies using natural killer T(NKT)cells, γδ T cells, mucosal-associated invariant T(MAIT)cells, and macrophages.

Generation of highly proliferative, rejuvenated cytotoxic T cell clones through pluripotency reprogramming for adoptive immunotherapy.

Adoptive immunotherapy has emerged as a powerful approach to cure cancer and chronic infections. Currently, the generation of a massive number of T cells that provide long-lasting immunity is challenged by exhaustion and differentiation-associated senescence, which inevitably arise during in vitro cloning and expansion. To circumvent these problems, several studies have proposed an induced pluripotent stem cell (iPSC)-mediated rejuvenation strategy to revitalize the exhausted/senescent T cell clones. Because iPSC-derived cytotoxic T lymphocytes (iPSC-CTLs) generated via commonly used monolayer systems have unfavorable, innate-like features such as aberrant natural killer (NK) activity and limited replication potential, we modified the redifferentiation culture to generate CD8αß+CD5+CCR7+CD45RA+CD56--adaptive iPSC-CTLs. The modified iPSC-CTLs exhibited early memory phenotype, including high replicative capacity and the ability to give rise to potent effector cells. In expansion culture with an optimized cytokine cocktail, iPSC-CTLs proliferated more than 1015-fold in a feeder-free condition. Our redifferentiation and expansion package of early memory iPSC-CTLs could supply memory and effector T cells for both autologous and allogeneic immunotherapies.

[Development of antigen-receptor modified allogeneic T cell from iPS cells for cancer immunotherapy].

The efficacy of T-cell therapy depends on the maintenance of antigen specificity, memory phenotype, longterm viability, and proliferative capacity of T cells in vivo. Personalized autologous T-cell therapies pose a few manufacturing challenges, in terms of quality, and supply stability. Recently, it has become possible to derive CD8 killer T cells from induced pluripotent stem cells (iPSCs) and develop CAR-CD8 killer T cells from allogeneic iPSCs. This article reviews CD8 killer T-cell induction from iPSCs, attempts to enhance process safety and reliability, and discusses the use of gene-editing technology for reducing allogeneic antigenicity.

[Development of CAR-T cell therapy using allogeneic iPS cells].

CAR-T therapy has shown excellent therapeutic efficacy in B-cell malignancy. Nevertheless, manufacturing stability, quality control, and CAR T-cell availability are still challenging because current CAR T-cell therapy is a personalized product derived from patient peripheral T-cells. However, allogeneic T-cells have emerged as a novel source to overcome this issue. Because induced pluripotent stem (iPS) cells are pluripotent stem cells derived from somatic cells and have in vitro self-renewal ability and pluripotency, they are expected to be a source of many regenerative medicinal products. Recently, it has become possible to generate CD8 killer T cells from iPS cells, and efforts have been made to generate CAR-CD8 killer T-cells from allogeneic iPS cells. This review discusses the induction of CD8 killer T-cells from iPS cells, efforts to improve the safety and certainty of the induction process for clinical use, and the utility of gene editing to reduce allogeneic antigenicity of iPS T-cells.

Non-clinical efficacy, safety and stable clinical cell processing of induced pluripotent stem cell-derived anti-glypican-3 chimeric antigen receptor-expressing natural killer/innate lymphoid cells.

The use of allogeneic, pluripotent stem-cell-derived immune cells for cancer immunotherapy has been the subject of recent clinical trials. In Japan, investigator-initiated clinical trials will soon begin for ovarian cancer treatment using human leukocyte antigen (HLA)-homozygous-induced pluripotent stem cell (iPSC)-derived anti-glypican-3 (GPC3) chimeric antigen receptor (CAR)-expressing natural killer/innate lymphoid cells (NK/ILC). Using pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, in terms of efficacy, safety and producibility. In this paper, we describe our methods for the stable, feeder-free production of CAR-expressing NK/ILC cells from CAR-transduced iPSC with clinically relevant scale and materials. The average number of cells that could be differentiated from 1.8-3.6 × 106 iPSC within 7 weeks was 1.8-4.0 × 109 . These cells showed stable CD45/CD7/CAR expression, effector functions of cytotoxicity and interferon gamma (IFN-γ) production against GPC3-expressing tumor cells. When the CAR-NK/ILC cells were injected into a GPC3-positive, ovarian-tumor-bearing, immunodeficient mouse model, we observed a significant therapeutic effect that prolonged the survival of the animals. When the cells were injected into immunodeficient mice during non-clinical safety tests, no acute systemic toxicity or tumorigenicity of the final product or residual iPSC was observed. In addition, our test results for the CAR-NK/ILC cells generated with clinical manufacturing standards are encouraging, and these methods should accelerate the development of allogeneic pluripotent stem cell-based immune cell cancer therapies.

Reprogramming away from the exhausted T cell state.

Induced pluripotent stem cells (iPSCs) describe somatic cells that have been reprogrammed to the pluripotent state from which they can then be differentiated into any cell type of the body. This ability has tremendous implications on a wide number of medical sciences and applications, including cancer treatments. In many cancer patients, tumor infiltrating lymphocytes (TILs) have reached an exhausted state and are unable to exert effector function despite detecting and localizing at the tumor. Although the isolation, ex vivo expansion and transplantation of TILs is effective in a significant group of patients, too many patients do not respond positively to this treatment, in part because the expanded TIL population does not include a sufficient number of cells with the naïve or memory phenotype. Cell reprogramming using iPSC technologies aims to overcome this problem by returning TILs to the pluripotent state from which they can be differentiated into a heterogeneous population of T cells that are best suited to combat the tumor.

Toward the development of true "off-the-shelf" synthetic T-cell immunotherapy.

Recent outstanding clinical results produced by engineered T cells, including chimeric antigen receptors, have already facilitated further research that broadens their applicability. One such direction is to explore new T cell sources for allogeneic "off-the-shelf" adoptive immunotherapy. Human pluripotent stem cells could serve as an alternative cell source for this purpose due to their unique features of infinite propagation ability and pluripotency. Here, we describe the current state of engineered T cell transfer with the focus on cell manufacturing processes and the potentials and challenges of induced pluripotent stem cell-derived T cells as a starting material to construct off-the-shelf T-cell banks.

Repurposing the Cord Blood Bank for Haplobanking of HLA-Homozygous iPSCs and Their Usefulness to Multiple Populations.

Although autologous induced pluripotent stem cells (iPSCs) can potentially be useful for treating patients without immune rejection, in reality it will be extremely expensive and labor-intensive to make iPSCs to realize personalized medicine. An alternative approach is to make use of human leukocyte antigen (HLA) haplotype homozygous donors to provide HLA matched iPSC products to significant numbers of patients. To establish a haplobank of iPSCs, we repurposed the cord blood bank by screening ∼4,200 high resolution HLA typed cord blood samples, and selected those homozygous for the 10 most frequent HLA-A,-B,-DRB1 haplotypes in the Korean population. Following the generation of 10 iPSC lines, we conducted a comprehensive characterization, including morphology, expression of pluripotent markers and cell surface antigens, three-germ layer formation, vector clearance, mycoplasma/microbiological/viral contamination, endotoxin, and short tandem repeat (STR) assays. Various genomic analyses using microarray and comparative genomic hybridization (aCGH)-based single nucleotide polymorphism (SNP) and copy number variation (CNV) were also conducted. These 10 HLA-homozygous iPSC lines match 41.07% of the Korean population. Comparative analysis of HLA population data shows that they are also of use in other Asian populations, such as Japan, with some limited utility in ethnically diverse populations, such as the UK. Taken together, the generation of the 10 most frequent Korean HLA-homozygous iPSC lines serves as a useful pointer for the development of optimal methods for iPSC generation and quality control and indicates the benefits and limitations of collaborative HLA driven selection of donors for future stocking of worldwide iPSC haplobanks. Stem Cells 2018;36:1552-1566.

Human AK2 links intracellular bioenergetic redistribution to the fate of hematopoietic progenitors.

AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells.

[Immune cell therapy using iPS cells].

Adoptive cell therapy using tumor-infiltrating T cells has shown durable responses in patients with melanoma, and immunotherapy using genetically engineered T cells (TCR-T or CAR-T) is rapidly emerging as a promising treatment, especially for hematological malignancies. However, the progress is limited because of the lack of readily available good-quality human T cells. Although the efficacy of adoptive cell therapy correlates with the quality of infusing T cells, most antigen-specific T cells in patients with cancer have been exhausted. To overcome this, we have reprogrammed donor (or original) T cells to iPS cells (T-iPS) and differentiated these into rejuvenated antigen-specific cells (T-iPS-T). Moreover, iPS cells provide an unlimited source of genetically engineered T cells such as TCR/CAR-T or PD-1 knockout T cells. The iPS cells' potential for immune cell therapy is infinite.

T-cell-restricted T-bet overexpression induces aberrant hematopoiesis of myeloid cells and impairs function of macrophages in the lung.

Although overexpression of T-bet, a master transcription factor in type-1 helper T lymphocytes, has been reported in several hematologic and immune diseases, its role in their pathogenesis is not fully understood. In the present study, we used transgenic model mice (T-bet(tg/wt) and T-bet(tg/tg)) to investigate the effects of T-bet overexpression selectively in T lymphocytes on the development of hematologic and immune diseases. The results showed that T-bet overexpression in T cells spontaneously induced maturation arrest in the mononuclear phagocyte lineage, as well as spontaneous dermatitis and pulmonary alveolar proteinosis (PAP)-like disease in T-bet(tg/wt) and T-bet(tg/tg) mice, respectively. T-bet(tg/tg) alveoli with the PAP phenotype showed remarkable reorganization of alveolar mononuclear phagocyte subpopulations and impaired function, in addition to augmented T-cell infiltration. In addition, PAP development in T-bet(tg/tg) mice was found to be associated with increased migration of myeloid cells from the bone marrow into the peripheral blood. These findings reveal an unexpected link between T-bet overexpression in T lymphocytes and the development of PAP caused by reorganization of mononuclear phagocytes in the lung, and provide new insight into the molecular pathogenesis of secondary PAP accompanied by hematologic disorders.

Immune cells derived from induced pluripotent stem cells: a new cell source for cancer immunotherapy.

Clinical results have shown promising therapeutic potential of engineered T cell express- ing TCR (T cell antigen receptor) or CAR (chimeric antigen receptor). However, high cost, donor cell availability, complicated procedure of genetic engineering prevent their wider ap- plication. iPSCs (induced pluripotent stem cells) are attractive source of immune cells includ- ing T cells and dendritic cells, and may provide an unlimited cell source for adoptive immune cell therapy. Genetic engineering of iPSCs may confer enhanced anti-tumor activity and ena- ble the escape from immune rejection. Here, we discuss the potential of iPSCs as a novel source of anti-tumor immune cells.

Genome editing iPSC to purposing enhancement of induce CD8 killer T cell function for regenerative immunotherapy.

In recent years, immunotherapy has become a standard cancer therapy, joining surgery, chemotherapy, and radiation therapy. This therapeutic approach involves the use of patient-derived antigen-specific T cells or genetically modified T cells engineered with chimeric antigen receptors (CAR) or T cell receptors (TCR) that specifically target cancer antigens. However, T cells require ex vivo stimulation for proliferation when used in therapy, and the resulting "exhaustion," which is characterized by a diminished proliferation capacity and anti-tumor activity, poses a significant challenge. As a solution, we reported "rejuvenated" CD8 + T cells that possess high proliferation capacity from induced pluripotent stem cells (iPSCs) in 2013. This review discusses the status and future developments in immunotherapy using iPSC-derived T cells, drawing insights from our research to overcome the exhaustion associated with antigen-specific T cell therapy.

Effective and stable gene transduction in rhesus macaque iPSCs capable of T-lineage differentiation utilizing the piggyBac system.

Introduction: Genetically modified human induced pluripotent stem cell (iPSC)-based regenerative medicine has substantial potential in the treatment of refractory human diseases. Thus, preclinical studies on the safety and efficacy of these products are essential. Non-human primate (NHP) models such as the rhesus macaque are highly similar to humans in terms of size, lifespan, and immune system, rendering them superior models. However, effective gene transduction in rhesus macaque iPSCs (Rh-iPSCs) remains challenging. In this study, we investigated the effective gene transduction into Rh-iPSCs and its effect on differentiation efficiency. Methods: We established a gene transduction method using the piggyBac transposon vector system. Gene transduced Rh-iPSCs were analyzed for undifferentiated markers. We did teratoma assay to check pluripotency. Gene transduced Rh-iPSCs were differentiated into hematopoietic stem and progenitor cells (HSPCs) and T-cell lineage cells. Additionally, gene transduced Rh-iPSCs were compared the differentiation efficiency with parental Rh-iPSCs. Results: We could establish a gene transduction method using the piggyBac transposon vector system, demonstrating high efficiency and stable transgene expression in Rh-iPSCs. These Rh-iPSCs maintained long-term gene expression while expressing undifferentiated markers. Teratoma assay indicated that these Rh-iPSCs had pluripotency. These Rh-iPSCs could differentiate into HPSCs and T cells that express transgenes. These Rh-iPSCs can differentiate into hematopoietic stem cells and T cells that express transgenes. No significant differences in efficiency of differentiation were observed between parental Rh-iPSCs and these Rh-iPSCs. Conclusions: These results indicate that the piggyBac transposon vector is an excellent gene transfer tool for rhesus macaque iPSCs and could contribute to the advancement of preclinical studies using rhesus macaque iPSCs.

Human iPSC-derived CD4+ Treg-like cells engineered with chimeric antigen receptors control GvHD in a xenograft model.

CD4+ T cells induced from human iPSCs (iCD4+ T cells) offer a therapeutic opportunity for overcoming immune pathologies arising from hematopoietic stem cell transplantation. However, most iCD4+ T cells are conventional helper T cells, which secrete inflammatory cytokines. We induced high-level expression of FOXP3, a master transcription factor of regulatory T cells, in iCD4+ T cells. Human iPSC-derived, FOXP3-induced CD4+ T (iCD4+ Treg-like) cells did not secrete inflammatory cytokines upon activation. Moreover, they showed demethylation of the Treg-specific demethylation region, suggesting successful conversion to immunosuppressive iCD4+ Treg-like cells. We further assessed these iCD4+ Treg-like cells for CAR-mediated immunosuppressive ability. HLA-A2 CAR-transduced iCD4+ Treg-like cells inhibited CD8+ cytotoxic T cell (CTL) division in a mixed lymphocyte reaction assay with A2+ allogeneic CTLs and suppressed xenogeneic graft-versus-host disease (GVHD) in NSG mice treated with A2+ human PBMCs. In most cases, these cells suppressed the xenogeneic GvHD progression as much as natural CD25+CD127- Tregs did.

A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism.

Memory T cells demonstrate superior in vivo persistence and antitumor efficacy. However, methods for manufacturing less differentiated T cells are not yet well-established. Here, we show that producing chimeric antigen receptor (CAR)-T cells using berbamine (BBM), a natural compound found in the Chinese herbal medicine Berberis amurensis, enhances the antitumor efficacy of CAR-T cells. BBM is identified through cell-based screening of chemical compounds using induced pluripotent stem cell-derived T cells, leading to improved viability with a memory T cell phenotype. Transcriptomics and metabolomics using stem cell memory T cells reveal that BBM broadly enhances lipid metabolism. Furthermore, the addition of BBM downregulates the phosphorylation of p38 mitogen-activated protein kinase and enhanced mitochondrial respiration. CD19-CAR-T cells cultured with BBM also extend the survival of leukaemia mouse models due to their superior in vivo persistence. This technology offers a straightforward approach to enhancing the antitumor efficacy of CAR-T cells.