Current progress of CAR-T-cell therapy for patients with multiple myeloma.

Currently available chimeric antigen receptor (CAR)-engineered T-cell therapies targeting B-cell maturation antigen (BCMA), namely, idecabtagene vicleucel and ciltacabtagene autoleucel, have shown marked efficacy against relapsed and refractory multiple myeloma. However, further improvement in CAR-T-cell function is warranted as most patients treated with these products eventually relapse due to various mechanisms such as antigen loss and T-cell dysfunction or disappearance. Strategies for improving CAR-T-cell function include targeting of dual antigens, enhancing cell longevity through genetic modification, and eliminating the immunosuppressive tumor microenvironment. Serious side effects can also occur after CAR-T-cell infusions. Although understanding of the molecular pathogenesis of cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome is growing, the unique movement disorder caused by BCMA-targeted therapy is less understood, and its molecular mechanisms must be further elucidated to establish better management strategies. In this article, we will review the current status of BCMA-targeting CAR-T-cell therapy. We will also highlight progress in the development of CAR-T cells targeting other antigens, as well as universal allogeneic CAR-T cells and bispecific antibodies.

Development of a chimeric cytokine receptor that captures IL-6 and enhances the antitumor response of CAR-T cells.

The efficacy of chimeric antigen receptor (CAR)-engineered T cell therapy is suboptimal in most cancers, necessitating further improvement in their therapeutic actions. However, enhancing antitumor T cell response inevitably confers an increased risk of cytokine release syndrome associated with monocyte-derived interleukin-6 (IL-6). Thus, an approach to simultaneously enhance therapeutic efficacy and safety is warranted. Here, we develop a chimeric cytokine receptor composed of the extracellular domains of GP130 and IL6RA linked to the transmembrane and cytoplasmic domain of IL-7R mutant that constitutively activates the JAK-STAT pathway (G6/7R or G6/7R-M452L). CAR-T cells with G6/7R efficiently absorb and degrade monocyte-derived IL-6 in vitro. The G6/7R-expressing CAR-T cells show superior expansion and persistence in vivo, resulting in durable antitumor response in both liquid and solid tumor mouse models. Our strategy can be widely applicable to CAR-T cell therapy to enhance its efficacy and safety, irrespective of the target antigen.

High CD62L expression predicts the generation of chimeric antigen receptor T cells with potent effector functions.

The efficient generation of chimeric antigen receptor (CAR) T cells is highly influenced by the quality of apheresed T cells. Healthy donor-derived T cells usually proliferate better than patients-derived T cells and are precious resources to generate off-the-shelf CAR-T cells. However, relatively little is known about the determinants that affect the efficient generation of CAR-T cells from healthy donor-derived peripheral blood mononuclear cells (PBMCs) compared with those from the patients' own PBMCs. We here examined the efficiency of CAR-T cell generation from multiple healthy donor samples and analyzed its association with the phenotypic features of the starting peripheral blood T cells. We found that CD62L expression levels within CD8+ T cells were significantly correlated with CAR-T cell expansion. Moreover, high CD62L expression within naïve T cells was associated with the efficient expansion of T cells with a stem cell-like memory phenotype, an indicator of high-quality infusion products. Intriguingly, genetic disruption of CD62L significantly impaired CAR-T cell proliferation and cytokine production upon antigen stimulation. Conversely, ectopic expression of a shedding-resistant CD62L mutant augmented CAR-T cell effector functions compared to unmodified CAR-T cells, resulting in improved antitumor activity in vivo. Collectively, we identified the surface expression of CD62L as a concise indicator of potent T-cell proliferation. CD62L expression is also associated with the functional properties of CAR-T cells. These findings are potentially applicable to selecting optimal donors to massively generate CAR-T cell products.

Gene editing technology to improve antitumor T-cell functions in adoptive immunotherapy.

Adoptive immunotherapy, in which tumor-reactive T cells are prepared in vitro for adoptive transfer to the patient, can induce an objective clinical response in specific types of cancer. In particular, chimeric antigen receptor (CAR)-redirected T-cell therapy has shown robust responses in hematologic malignancies. However, its efficacy against most of the other tumors is still insufficient, which remains an unmet medical need. Accumulating evidence suggests that modifying specific genes can enhance antitumor T-cell properties. Epigenetic factors have been particularly implicated in the remodeling of T-cell functions, including changes to dysfunctional states such as terminal differentiation and exhaustion. Genetic ablation of key epigenetic molecules prevents the dysfunctional reprogramming of T cells and preserves their functional properties.Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based gene editing is a valuable tool to enable efficient and specific gene editing in cultured T cells. A number of studies have already identified promising targets to improve the therapeutic efficacy of CAR-T cells using genome-wide or focused CRISPR screening. In this review, we will present recent representative findings on molecular insights into T-cell dysfunction and how genetic modification contributes to overcoming it. We will also discuss several technical advances to achieve efficient gene modification using the CRISPR and other novel platforms.

Cytokine signaling in chimeric antigen receptor T-cell therapy.

Adoptive immunotherapy using chimeric antigen-receptor (CAR)-engineered T cells can induce robust antitumor responses against hematologic malignancies. However, its efficacy is not durable in the majority of the patients, warranting further improvement of T-cell functions. Cytokine signaling is one of the key cascades regulating T-cell survival and effector functions. In addition to cytokines that use the common γ chain as a receptor subunit, multiple cytokines regulate T-cell functions directly or indirectly. Modulating cytokine signaling in CAR-T cells by genetic engineering is one promising strategy to augment their therapeutic efficacy. These strategies include ectopic expression of cytokines, cytokine receptors, and synthetic molecules that mimic endogenous cytokine signaling. Alternatively, autocrine IL-2 signaling can be augmented through reprogramming of CAR-T cell properties through transcriptional and epigenetic modification. On the other hand, cytokine production by CAR-T cells triggers systemic inflammatory responses, which mainly manifest as adverse events such as cytokine-release syndrome (CRS) and neurotoxicity. In addition to inhibiting direct inflammatory mediators such as IL-6 and IL-1 released from activated macrophages, suppression of T-cell-derived cytokines associated with the priming of macrophages can be accomplished through genetic modification of CAR-T cells. In this review, I will outline recently developed synthetic biology approaches to exploit cytokine signaling to enhance CAR-T cell functions. I will also discuss therapeutic target molecules to prevent or alleviate CAR-T cell-related toxicities.

Epigenetic profiles guide improved CRISPR/Cas9-mediated gene knockout in human T cells.

Genetic modification of specific genes is emerging as a useful tool to enhance the functions of antitumor T cells in adoptive immunotherapy. Current advances in CRISPR/Cas9 technology enable gene knockout during in vitro preparation of infused T-cell products through transient transfection of a Cas9-guide RNA (gRNA) ribonucleoprotein complex. However, selecting optimal gRNAs remains a major challenge for efficient gene ablation. Although multiple in silico tools to predict the targeting efficiency have been developed, their performance has not been validated in cultured human T cells. Here, we explored a strategy to select optimal gRNAs using our pooled data on CRISPR/Cas9-mediated gene knockout in human T cells. The currently available prediction tools alone were insufficient to accurately predict the indel percentage in T cells. We used data on the epigenetic profiles of cultured T cells obtained from transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Combining the epigenetic information with sequence-based prediction tools significantly improved the gene-editing efficiency. We further demonstrate that epigenetically closed regions can be targeted by designing two gRNAs in adjacent regions. Finally, we demonstrate that the gene-editing efficiency of unstimulated T cells can be enhanced through pretreatment with IL-7. These findings enable more efficient gene editing in human T cells.

AKT2 inhibition accelerates the acquisition of phagocytic ability in induced pluripotent stem cell-derived neutrophils.

Neutrophils are key components of the immune system that inhibit bacterial infections. Systemic bacterial infections can cause lethal conditions, especially in patients with neutropenia associated with chemotherapy or other systemic illnesses; hence, early detection of the symptoms and prompt management are crucial in such cases. Previously, we established expandable engineered neutrophil-primed progenitors (NeuPs-XL) using human-induced pluripotent stem cells (iPSCs), which can produce neutrophil-like cells at a clinically suitable scale within 4 days of inducing myeloid differentiation. In this study, using small-molecule compound-based screening, we detected that MK-2206, a selective pan-AKT inhibitor, can accelerate this differentiation process, promote phagocytic ability in neutrophils, and enhance cytokine and chemokine expression in response to lipopolysaccharides. The inhibition of AKT2 has been identified as the key mechanism underlying this acceleration. These results can make a substantial contribution to the development of strategies for the prompt production of clinically applicable iPSC-derived neutrophils, which can potentially lead to the management of severe infections associated with life-threatening neutropenia and the effective treatment of related health conditions in the future.

CD83 expression characterizes precursor exhausted T cell population.

T cell exhaustion is a main obstacle against effective cancer immunotherapy. Exhausted T cells include a subpopulation that maintains proliferative capacity, referred to as precursor exhausted T cells (TPEX). While functionally distinct and important for antitumor immunity, TPEX possess some overlapping phenotypic features with the other T-cell subsets within the heterogeneous tumor-infiltrating T-lymphocytes (TIL). Here we explore surface marker profiles unique to TPEX using the tumor models treated by chimeric antigen receptor (CAR)-engineered T cells. We find that CD83 is predominantly expressed in the CCR7+PD1+ intratumoral CAR-T cells compared with the CCR7-PD1+ (terminally differentiated) and CAR-negative (bystander) T cells. The CD83+CCR7+ CAR-T cells exhibit superior antigen-induced proliferation and IL-2 production compared with the CD83- T cells. Moreover, we confirm selective expression of CD83 in the CCR7+PD1+ T-cell population in primary TIL samples. Our findings identify CD83 as a marker to discriminate TPEX from terminally exhausted and bystander TIL.

[Development of next-generation chimeric antigen receptor-engineered T-cell therapy].

Chimeric antigen receptor (CAR)-engineered T-cell therapy against B-cell malignancies and multiple myeloma was recently introduced for clinical use. However, the efficacy of CAR-T cell therapy is not durable in most patients, warranting the development of CAR-T cells with additional genetic modification or engineering of synthetic molecules to enhance their functions. This review will provide an overview of the molecular mechanisms underlying the functional alteration of T cells, especially transcriptional networks associated with memory formation and T cell exhaustion. In addition, methods to rationally improve CAR-T cell functions based on these mechanistic insights will be discussed.

[Development of universal CAR-T cells].

The efficacy of adoptive immunotherapy using CD19-targeting chimeric antigen receptor (CAR)-engineered T cells against B-cell malignancies has already been established in the clinic. However, high economic costs and heterogeneous quality of CAR-T cells derived from individual patients hinder further expansion of their applicability to various cancer types, including solid tumors. Mass CAR-T cell production from healthy donors is a promising approach to overcome these problems, given that allogeneic immunity elicited against donor CAR-T cells by the recipient's immune system is controlled. CAR-T cells genetically ablated with T-cell receptor and human leukocyte antigen molecules, referred to as universal CAR-T cells, may enable the use of allogeneic T cells for off-the-shelf adoptive cancer immunotherapy. However, several concerns, such as poor persistence of infused CAR-T cells and chromosomal abnormalities due to genome editing, remain to be addressed. Thus, recent clinical trials on universal CAR-T cells are summarized and future perspectives to overcome current challenges are discussed in this review.

Epigenetic engineering for optimal chimeric antigen receptor T cell therapy.

Recent advancements in cancer immunotherapy, such as chimeric antigen receptor (CAR)-engineered T cell therapy and immune checkpoint therapy, have significantly improved the clinical outcomes of patients with several types of cancer. To broaden its applicability further and induce durable therapeutic efficacy, it is imperative to understand how antitumor T cells elicit cytotoxic functions, survive as memory T cells, or are impaired in their effector functions (exhausted) at the molecular level. T cell properties are regulated by their gene expression profiles, which are further controlled by epigenetic architectures, such as DNA methylation and histone modifications. Multiple studies have elucidated specific epigenetic genes associated with T-cell phenotypic changes. Conversely, exogenous modification of these key epigenetic factors can significantly alter T cell functions by extensively altering the transcription network, which can be applied in cancer immunotherapy by improving T cell persistence or augmenting effector functions. As CAR-T cell therapy involves a genetic engineering step during the preparation of the infusion products, it would be a feasible strategy to additionally modulate specific epigenetic genes in CAR-T cells to improve their quality. Here, we review recent studies investigating how individual epigenetic factors play a crucial role in T-cell biology. We further discuss future directions to integrate these findings for optimal cancer immunotherapy.

[Molecular Profiles of Exhausted T Cells and Their Impact on Response to Immune Checkpoint Blockade].

T cell exhaustion is induced in the context of chronic virus infection and tumor microenvironment, in which cytotoxic T cells are repeatedly exposed to the target antigen and deprived of their effector functions. Multiple studies have already shown the significant impact of immune checkpoint molecules such as PD1 on functional properties of exhausted T cells. In addition to these signals, exhausted T cells possess distinct transcriptional and epigenetic profiles compared with conventional effector and memory T cells. Importantly, most of these features are not affected by immune checkpoint blockade, suggesting that genetic and epigenetic remodeling of T cells is an underlying molecular mechanism essential for T cell exhaustion. Moreover, it has now been evident that exhausted T cells are a heterogeneous cell population composed of distinct T cell subsets, and these functional differences profoundly affect therapeutic efficacy of cancer immunotherapy. In this review, I will discuss recent studies investigating molecular mechanisms of T cell exhaustion, including novel key molecules essentially associated with T cell exhaustion. These findings are potentially applicable to reinvigorate effector functions of exhausted T cells.

Dissecting the heterogeneity of exhausted T cells at the molecular level.

Our understanding of mechanisms underlying T-cell exhaustion has been refined by analysis of exhausted T cells at the molecular level. The development and functions of exhausted T cells are regulated by a number of transcription factors, epigenetic factors and metabolic enzymes. In addition, recent work to dissect exhausted T cells at the single-cell level has enabled us to discover a precursor exhausted T-cell subset equipped with long-term survival capacity. Starting from the analysis of mouse models, the existence of precursor exhausted T cells has also been documented in human T cells in the context of chronic virus infections or tumors. Clinical data suggest that evaluating the quality of exhausted T cells on the basis of their differentiation status may be helpful to predict the therapeutic response to inhibition of programmed death 1 (PD1). Moreover, beyond immune-checkpoint blockade, novel therapeutic approaches to re-invigorate exhausted T cells have been explored based on molecular insights into T-cell exhaustion. Here I will discuss key molecular profiles associated with the development, maintenance and differentiation of exhausted T cells and how these findings can be applicable in the field of cancer immunotherapy.

Genetic ablation of PRDM1 in antitumor T cells enhances therapeutic efficacy of adoptive immunotherapy.

Adoptive cancer immunotherapy can induce objective clinical efficacy in patients with advanced cancer; however, a sustained response is achieved in a minority of cases. The persistence of infused T cells is an essential determinant of a durable therapeutic response. Antitumor T cells undergo a genome-wide remodeling of the epigenetic architecture upon repeated antigen encounters, which inevitably induces progressive T-cell differentiation and the loss of longevity. In this study, we identified PR domain zinc finger protein 1 (PRDM1) ie, Blimp-1, as a key epigenetic gene associated with terminal T-cell differentiation. The genetic knockout of PRDM1 by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) supported the maintenance of an early memory phenotype and polyfunctional cytokine secretion in repeatedly stimulated chimeric antigen receptor (CAR)-engineered T cells. PRDM1 disruption promoted the expansion of less differentiated memory CAR-T cells in vivo, which enhanced T-cell persistence and improved therapeutic efficacy in multiple tumor models. Mechanistically, PRDM1-ablated T cells displayed enhanced chromatin accessibility of the genes that regulate memory formation, thereby leading to the acquisition of gene expression profiles representative of early memory T cells. PRDM1 knockout also facilitated maintaining an early memory phenotype and cytokine polyfunctionality in T-cell receptor-engineered T cells as well as tumor-infiltrating lymphocytes. In other words, targeting PRDM1 enabled the generation of superior antitumor T cells, which is potentially applicable to a wide range of adoptive cancer immunotherapies.

CAMK2G is identified as a novel therapeutic target for myelofibrosis.

Although JAK1/2 inhibition is effective in alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here, we established induced pluripotent stem cells (iPSCs) derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were used for chemical screening, and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutation recurrently detected in MF patients, were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 γ subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515L decreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes, such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.

CD62L expression level determines the cell fate of myeloid progenitors.

Hematopoietic cells differentiate through several progenitors in a hierarchical manner, and recent single-cell analyses have revealed substantial heterogeneity within each progenitor. Although common myeloid progenitors (CMPs) are defined as a multipotent cell population that can differentiate into granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythrocyte progenitors (MEPs), and GMPs generate neutrophils and monocytes, these myeloid progenitors must contain some lineage-committed progenitors. Through gene expression analysis at single-cell levels, we identified CD62L as a marker to reveal the heterogeneity. We confirmed that CD62L-negative CMPs represent "bona fide" CMPs, whereas CD62L-high CMPs are mostly restricted to GMP potentials both in mice and humans. In addition, we identified CD62L-negative GMPs as the most immature subsets in GMPs and Ly6C+CD62L-intermediate and Ly6C+CD62L-high GMPs are skewed to neutrophil and monocyte differentiation in mice, respectively. Our findings contribute to more profound understanding about the mechanism of myeloid differentiation.

Efficient production of human neutrophils from iPSCs that prevent murine lethal infection with immune cell recruitment.

Neutrophils play an essential role in innate immune responses to bacterial and fungal infections, and loss of neutrophil function can increase the risk of acquiring lethal infections in clinical settings. Here, we show that engineered neutrophil-primed progenitors derived from human induced pluripotent stem cells can produce functional neutrophil-like cells at a clinically applicable scale that can act rapidly in vivo against lethal bacterial infections. Using 5 different mouse models, we systematically demonstrated that these neutrophil-like cells migrate to sites of inflammation and infection and increase survival against bacterial infection. In addition, we found that these human neutrophil-like cells can recruit murine immune cells. This system potentially provides a straight-forward solution for patients with neutrophil deficiency: an off-the-shelf neutrophil transfusion. This platform should facilitate the administration of human neutrophils for a broad spectrum of physiological and pathological conditions.

Evi1 upregulates Fbp1 and supports progression of acute myeloid leukemia through pentose phosphate pathway activation.

Evi1 is a transcription factor essential for the development as well as progression of acute myeloid leukemia (AML) and high Evi1 AML is associated with extremely poor clinical outcome. Since targeting metabolic vulnerability is the emerging therapeutic strategy of cancer, we herein investigated a novel therapeutic target of Evi1 by analyzing transcriptomic, epigenetic, and metabolomic profiling of mouse high Evi1 leukemia cells. We revealed that Evi1 overexpression and Evi1-driven leukemic transformation upregulate transcription of gluconeogenesis enzyme Fbp1 and other pentose phosphate enzymes with interaction between Evi1 and the enhancer region of these genes. Metabolome analysis using Evi1-overexpressing leukemia cells uncovered pentose phosphate pathway upregulation by Evi1 overexpression. Suppression of Fbp1 as well as pentose phosphate pathway enzymes by shRNA-mediated knockdown selectively decreased Evi1-driven leukemogenesis in vitro. Moreover, pharmacological or shRNA-mediated Fbp1 inhibition in secondarily transplanted Evi1-overexpressing leukemia mouse significantly decreased leukemia cell burden. Collectively, targeting FBP1 is a promising therapeutic strategy of high Evi1 AML.

Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4+ T cells.

Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses.

Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.

Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.