論文

造血器悪性腫瘍である急性骨髄性白血病では，正常造血系と類似して白血病幹細胞(leukemia initiating cell, LIC)を頂点とした階層構造が存在すると考えられている。治療抵抗性・再発の原因となるLICの特性を知ることが今後の新規治療法開発にとり重要であるが，とりわけ転写因子NF-κBは近年多くの白血病症例でその恒常的活性化が報告されており，治療標的として注目されている。興味深いことに，LICはTNF-αの自己分泌によりNF-κBの恒常的活性化を維持しており，逆に活性化されたNF-κBはTNF-αの高発現を維持することで，両者はポジティブ・フィードバックを形成している。マウス白血病モデルにおいて，TNF-α, NF-κBを白血病細胞において欠損させると白血病の進展が著しく阻害されることから，両者がLICの維持，増殖に重要な役割を果たしていることがわかる。さらにTNF-αによるNF-κBの十分な活性化にはプロテアソームの働きが必要であり，これらはいずれも有望な治療標的であることから，将来的な臨床応用が期待される。

キメラ抗原受容体（chimeric antigen receptor, CAR）導入T細胞療法は，B細胞性腫瘍や多発性骨髄腫などの造血器腫瘍に対する実用化が近年進んでいる。一方で再発率が高く，多くの症例で一過性の効果にとどまるのが現状で，持続的な効果を誘導するために様々な遺伝子改変や人工遺伝子導入を行ったCAR-T細胞の開発が行われている。本総説ではT細胞機能に関わる分子メカニズム，特にメモリー形成や疲弊誘導に伴う転写ネットワーク機構に触れながら，それらの理解に基づくCAR-T細胞の機能改変例を紹介する。

キメラ抗原受容体（chimeric antigen receptor, CAR）導入T細胞療法は，既に実臨床で用いられているCD19を標的としたB細胞性腫瘍に対する治療から，今後は固形がんに対しても適応拡大が期待される。その上で自己T細胞から個別にCAR-T細胞を製造することに関わるコスト，T細胞の質のばらつきなどが問題となっており，これを解決するために他家由来，すなわち健常人ドナーから大量にCAR-T細胞製造して使用する，“universal CAR-T cell”が注目されている。Universal CAR-T細胞療法を標準治療として確立する上で，治療効果・安全性の検証が臨床試験において進められている。特にドナー・レシピエント双方向のアロ免疫反応を抑制することがポイントとなり，主に遺伝子改変による免疫原性の減弱がはかられているが，これまでの臨床試験では十分な効果が確認されていない。またゲノム編集に伴う染色体異常が生じることが報告されており，安全性の確立も今後の課題であり，これらについて本稿で概説する。

T細胞に対する二重特異性抗体（BsAb）はCD3抗体を介して抗腫瘍活性をT細胞に賦与するが，長期的な治療効果は未だ不十分である。T細胞に最適な抗腫瘍効果を誘導するには複合的なシグナルが必要であり，我々はBsAb，共刺激分子，サイトカインなどの分子群を同時に表面に搭載したナノサイズの細胞膜由来膜小胞の作製技術を確立した。B細胞性造血器腫瘍に対し，CD19を標的としたBsAbに加え，共刺激分子（CD80，4-1BBL），T細胞の増殖・長期生存に関わるサイトカイン（IL-7，IL-15）を搭載した膜小胞は，T細胞に抗原特異的な抗腫瘍活性を効率よく誘導した。また抗PDL1抗体，CTLA4抗体，IL-12，IL-18など腫瘍微小環境に作用する分子を搭載し，腫瘍内マクロファージの改変や免疫抑制シグナルの解除を行えることを示した。この手法は従来の抗体医薬と比較して多面的な免疫制御シグナルを誘導できる。

Tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive immunotherapy potentially applicable to many types of solid tumors. Although gynecologic malignancies are promising targets for TIL therapy, its objective efficacy has not been established. Current TIL culture typically involves incubation of dissociated samples with high-dose IL-2 (HD-IL2) for weeks to enrich tumor-reactive T cells. While this protocol has been successfully used for melanoma TIL, it has not necessarily been optimized for other cancers. Here we investigated the method of efficiently expanding TILs derived from patients with gynecological cancers. TILs were incubated with HD-IL2 (HD-IL2-TILs) or stimulated with K562 cells expressing anti-CD3 mAb and CD80 (αCD3/CD80-TILs). We found that the αCD3/CD80-TILs showed significantly better proliferation than HD-IL2-TILs. The TIL populations that predominantly expanded upon αCD3/CD80 stimulation expressed high levels of PD-1 and CD28. CD28 co-stimulation was essential to overcome PD-1-mediated signals for growth suppression. We also identified DUSP4 as a negative regulator of TIL proliferation by downregulating ERK phosphorylation. The αCD3/CD80-TILs were reactive to tumor cells as shown by IFN-γ secretion and CD107a expression. Moreover, the αCD3/CD80-TILs were efficiently transduced with a chimeric cytokine receptor that we had previously developed to provide constitutive IL-7 signaling, resulting in superior in vivo persistence and antitumor effects without exogenous cytokine support in mouse models. Collectively, this study shows that direct stimulation of TILs with anti-CD3 mAb and CD28 co-stimulation achieves efficient expansion of tumor-reactive TILs. Genetic engineering of cytokine signaling in TILs may further enhance TIL functions and replace cytokine administration after TIL infusion.

Background While chimeric antigen receptor (CAR)-T cell therapy exhibits a robust therapeutic efficacy against B-cell malignancies and multiple myeloma, its efficacy and safety have not been established for acute myeloid leukemia (AML) and solid tumors due to the paucity of established target antigens. Some AML and solid tumor cells express tumor necrosis factor (TNF), which is initially expressed on the cell surface prior to shedding. Methods In this study, we obtained monoclonal antibodies against the N-terminal fragment of TNF (TNF-NTF) that remains on the cell surface after shedding. We then generated CAR-T cells to target TNF-NTF using the antibody sequence. To enhance the therapeutic efficacy of TNF-NTF CAR-T cells, we further engineered the previously developed chimeric cytokine receptor consisting of GP130, IL6R, and constitutively active IL7R with the M452L mutation (G6/7R). Results TNF-NTF CAR-T cells efficiently lysed TNF-expressing leukemia cells in vitro, while showing limited antitumor efficacy in vivo due to poor expansion and persistence. Activated T cells upregulate TNF, which was recognized by TNF-NTF CAR-T cells and led to fratricide. Genetic knockout (KO) of TNF significantly enhanced the viability and proliferation of TNF-NTF CAR-T cells, while slightly reducing their cytotoxic activity. In addition, ectopic expression of G6/7R improved the effector function of TNF-NTF CAR-T cells through constitutive activation of janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling. The G6/7R-expressing TNFKO TNF-NTF CAR-T cells exhibited superior persistence and durable antileukemic efficacy in vivo compared with parental CAR-T cells. We also confirmed that TNF-NTF CAR-T cells can target primary AML cells, including a leukemia-initiating population with colony-forming capacity. Unlike CD33, targeting TNF-NTF did not show cytotoxicity against normal hematopoietic stem/progenitor cells. Finally, we demonstrated the curative efficacy of G6/7R TNF-KO TNF-NTF CAR-T cells against TNF-expressing ovarian tumor cells in vivo. Conclusions Our studies highlight TNF-NTF as a promising cell surface target for CAR-T cell therapy that can be applied to AML as well as solid tumors.

Providing cytokine signaling is a key strategy to boost the efficacy of chimeric antigen receptor (CAR) T cell therapy. However, the individual roles of key downstream mediators, STAT3 and STAT5, remain incompletely understood. In this study, we engineered CAR T cells to express constitutively active mutants of STAT3 (Y640F; caSTAT3) and STAT5 (N642H; caSTAT5) to investigate their individual functions. In vitro, caSTAT3 CAR T cells exhibited enhanced effector function and a robust memory phenotype, with broader transcriptional changes involving both effector and memory associated genes compared to caSTAT5 CAR T cells. However, caSTAT3 CAR T cells failed to expand due to activation of apoptosis-related gene programs. In contrast, caSTAT5 CAR T cells demonstrated sustained proliferation over time. Despite the limited in vitro expansion, caSTAT3 CAR T cells exhibited reduced transgene toxicity in vivo and exerted durable antitumor activity in both leukemia and solid tumor models without significant off-tumor toxicity. Titrated expression of caSTAT3 maintained enhanced effector function without inducing apoptosis. On the other hand, caSTAT5 CAR T cells efficiently accumulated in tumors but also infiltrated non-tumor tissues, causing lethal systemic toxicity. Coexpression of caSTAT3 and caSTAT5 markedly enhanced the long-term proliferative capacity of CAR T cells, even in the absence of antigen stimulation or cytokine supplementation. These findings elucidate the distinct impacts of STAT3 and STAT5 activation on CAR T cell behavior and suggest that selective activation of STAT3 at optimal levels may improve CAR T cell efficacy while minimizing off-tumor toxicities.

キメラ抗原受容体（CAR）導入T細胞療法はB細胞性腫瘍，多発性骨髄腫に対して高い初回反応性を示すものの，再発率が高い。治療効果を改善する目的で，エピジェネティック因子やサイトカインシグナルに着目した遺伝子改変により，CAR-T細胞の長期生存能や疲弊への耐性を付与する研究開発が盛んである。また，治療効果に比例して発症リスクが高まるサイトカイン放出症候群などの治療毒性についても，予防・軽減するための研究開発が進んでいる。さらに，CAR-T細胞療法後のT細胞性腫瘍の発症リスクについて腫瘍発症に関わる要因を含めてまとまった報告が出てきており，長期的な安全性についての関心がますます高まっている。本稿ではこれらの観点について，最近の研究成果を概括しながら今後の開発に向けた展望について述べる。

キメラ抗原受容体（CAR）導入T細胞療法は造血器腫瘍に対して高い治療効果を示しているが，未だ長期的な治療効果は不十分であり，加えて固形腫瘍に対する奏効率は極めて限定的で，さらなる改良が求められている。サイトカインシグナルはCAR-T細胞の腫瘍制御能や副作用の発症に深く関与しており，このシグナルを改変することによってCAR-T細胞療法の治療効果や安全性を向上させることが数多く報告されている。本稿ではこれらの研究開発について代表的なものを紹介する。

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.

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.

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.

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.

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.

Background A number of immunotherapeutic approaches have been developed and are entering the clinic. Bispecific antibodies (BsAbs) are one of these modalities and induce robust efficacy by endogenous T cells in several hematological malignancies. However, most of the treated patients experience only a temporary benefit. Currently available BsAbs provide only anti-CD3 antibody-mediated T-cell stimulation, but not the costimulation or cytokine signaling essential for full T-cell activation. Here, we hypothesized that the simultaneous input of more comprehensive signals would elicit more robust and durable effector T-cell functions. Methods We genetically engineered the leukemia cell line K562 to express BsAbs, costimulatory ligands, cytokines, and blocking antibodies against immune checkpoint molecules on the cell surface, from which we obtained plasma membrane fractions by mechanical homogenization and subsequent isolation steps. Plasma membranes were reconstituted on the poly (lactic-co-glycolic acid) surface to generate membrane-coated nanoparticles (NPs). Alternatively, nano-sized membrane vesicles (MVs) were generated by ultrasonic dispersion of the isolated membranes. The antitumor function of NPs and MVs loaded with various immunomodulatory factors was evaluated in vitro and in vivo. Results Both membrane-coated NPs and MVs induced BsAb-mediated antigen-specific cytotoxic activity in non-specific T cells, with MVs inducing a slightly better response in vivo. Importantly, T-cell activation was elicited only in the presence of target tumor cells, providing a safety advantage for clinical use. NPs and MVs expressing costimulatory molecules (CD80/4-1BBL) and cytokines (interleukin (IL)-7/IL-15) further enhanced effector T-cell function and induced therapeutic efficacy in vivo. In addition, MVs expressing immune checkpoint antibodies and inflammatory cytokines IL-12 and IL-18 induced objective antitumor responses in solid tumor models partly by converting immunosuppressive macrophages to proinflammatory phenotypes and inducing cytotoxic T-cell infiltration into the tumor. Finally, we showed that MVs were also engineered to activate natural killer (NK) cells by loading multiple ligands. MVs loaded with BsAbs, 4-1BBL, IL-15, and IL-21 induced NK-cell cytotoxic activity in an antigen-specific manner. Conclusions We developed antitumor NPs and MVs that efficiently induced antitumor immune responses in vivo by simultaneously delivering multiple immunostimulatory signals to endogenous T cells. This platform enables the delivery of desired combinations of antitumor immune signals into T cells and NK cells.

Chimeric antigen receptor (CAR) T cell therapy often causes serious toxicities, such as cytokine release syndrome (CRS), mainly due to interleukin-6 (IL-6) secreted by monocyte lineage cells. Here, we describe a protocol to generate anti-CD19 CAR T cells and quantify human monocyte-derived IL-6 cocultured with CAR T cells and target tumor cells in vitro. We further describe a protocol to generate a humanized mouse model and evaluate CAR T cell-associated plasma IL-6 levels in vivo. For complete details on the use and execution of this protocol, please refer to Yoshikawa et al.1

Chimeric antigen receptor(CAR)T-cell therapy has already been in the clinic due to its marked efficacy against several hematologic cancers. However, a significant number of patients relapse after a transient response, and the applicable diseases are still limited. Cytokine signaling is one of the most promising targets to enhance the therapeutic efficacy of CAR-T cells. At the same time, several cytokines are closely associated with toxicity, as exemplified by cytokine release syndrome. Here I will present representative findings on cytokines that have been shown to modulate CAR-T cell functions. I will also present our recent studies on how to simultaneously address efficacy and safety issues in CAR-T cell therapy. We will also briefly discuss CAR-NK cells, in which cytokines play an essential role.