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Chimeric antigen receptor (CAR) T cells have revolutionized treatment of B cell malignancies. However, enhancing the efficacy of engineered T cells without compromising their safety is warranted. The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) inhibits release of cytolytic enzymes from cytotoxic T lymphocytes. Here, we examined the potency of EBAG9 silencing for the improvement of adoptive T cell therapy. MicroRNA (miRNA)-mediated EBAG9 downregulation in transplanted cytolytic CD8+ T cells (CTLs) from immunized mice improved their cytolytic competence in a tumor model. In tolerant female recipient mice that received organ transplants, a minor histocompatibility antigen was turned into a rejection antigen by Ebag9 deletion, indicating an immune checkpoint function for EBAG9. Considerably fewer EBAG9-silenced human CAR T cells were needed for tumor growth control in a xenotransplantation model. Transcriptome profiling did not reveal additional risks regarding genotoxicity or aberrant differentiation. A single-step retrovirus transduction process links CAR or TCR expression with miRNA-mediated EBAG9 downregulation. Despite higher cytolytic efficacy, release of cytokines associated with cytokine release syndrome remains unaffected. Collectively, EBAG9 silencing enhances effector capacity of TCR- and CAR-engineered T cells, results in improved tumor eradication, facilitates efficient manufacturing, and decreases the therapeutic dose.

In therapeutic cancer vaccines, vaccine antigens must be efficiently delivered to the antigen-presenting cells (dendritic cells and macrophages) located in the lymphoid organs (lymph nodes and spleen) at the appropriate time to induce a potent antitumor immune response. Nanoparticle-based delivery systems in cancer immunotherapy are of great interest in recent year. We have developed a novel cancer vaccine that can use self-assembled polysaccharide nanogel of cholesteryl group-modified pullulan (CHP) as an antigen delivery system for clinical cancer immunotherapy for the first time. Additionally, we recently proposed a novel technology that uses CHP nanogels to regulate the function of tumor-associated macrophages, leading to an improvement in the tumor microenvironment. When combined with other immunotherapies, macrophage function modulation using CHP nanogels demonstrated a potent inhibitory effect against cancers resistant to immune checkpoint inhibition therapies. In this review, we discuss the applications of our unique drug nanodelivery system for CHP nanogels.

The aim of this study was to determine the efficacy and the biomarkers of the CHP-NY-ESO-1 vaccine complexed with full-length NY-ESO-1 protein and a cholesteryl pullulan (CHP) in patients with esophageal squamous cell carcinoma (ESCC) after surgery. We conducted a randomized phase II trial. Fifty-four patients with NY-ESO-1-expressing ESCC who underwent radical surgery following cisplatin/5-fluorouracil-based neoadjuvant chemotherapy were assigned to receive either CHP-NY-ESO-1 vaccination or observation as control. Six doses of CHP-NY-ESO-1 were administered subcutaneously once every two weeks, followed by nine more doses once every four weeks. The endpoints were disease-free survival (DFS) and safety. Exploratory analysis of tumor tissues using gene-expression profiles was also performed to seek the biomarker. As there were no serious adverse events in 27 vaccinated patients, we verified the safety of the vaccine. DFS in 2 years were 56.0% and 58.3% in the vaccine arm and in the control, respectively. Twenty-four of 25 patients showed NY-ESO-1-specific IgG responses after vaccination. Analysis of intra-cohort correlations among vaccinated patients revealed that 5% or greater expression of NY-ESO-1 was a favorable factor. Comprehensive analysis of gene expression profiles revealed that the expression of the gene encoding polymeric immunoglobulin receptor (PIGR) in tumors had a significantly favorable impact on outcomes in the vaccinated cohort. The high PIGR-expressing tumors that had higher NY-ESO-1-specific IgA response tended to have favorable prognosis. These results suggest that PIGR would play a major role in tumor immunity in an antigen-specific manner during NY-ESO-1 vaccinations. The IgA response may be relevant.

BackgroundBecause of the shortage of ideal cell surface antigens, the development of T-cell receptor (TCR)-engineered T cells (TCR-T) that target intracellular antigens such as NY-ESO-1 is a promising approach for treating patients with solid tumors. However, endogenous TCRs in vector-transduced T cells have been suggested to impair cell-surface expression of transduced TCR while generating mispaired TCRs that can become self-reactive.MethodsWe conducted a first-in-human phase I clinical trial with the TCR-transduced T-cell product (TBI-1301) in patients with NY-ESO-1-expressing solid tumors. In manufacturing TCR-T cells, we used a novel affinity-enhanced NY-ESO-1-specific TCR that was transduced by a retroviral vector that enables siRNA (small interfering RNA)-mediated silencing of endogenous TCR. The patients were divided into two cohorts. Cohort 1 was given a dose of 5×108 cells (whole cells including TCR-T cells) preconditioned with 1500 mg/m2 cyclophosphamide. Cohort 2 was given 5× 109 cells preconditioned with 1500 mg/m2 cyclophosphamide.ResultsIn vitro study showed that both the CD8+ and CD4+ T fractions of TCR-T cells exhibited cytotoxic effects against NY-ESO-1-expressing tumor cells. Three patients and six patients were allocated to cohort 1 and cohort 2, respectively. Three of the six patients who received 5×109 cells showed tumor response, while three patients developed early-onset cytokine release syndrome (CRS). One of the patients developed a grade 3 lung injury associated with the infiltration of the TCR-T cells. No siRNA-related adverse events other than CRS were observed. Cytokines including interleukin 6 I and monocyte chemotactic protein-1/chemokine (C-C motif) ligand (CCL2)increased in the sera of patients with CRS. In vitro analysis showed these cytokines were not secreted from the T cells infused. A significant fraction of the manufactured T cells in patients with CRS was found to express either CD244, CD39, or both at high levels.ConclusionsThe trial showed that endogenous TCR-silenced and affinity-enhanced NY-ESO-1 TCR-T cells were safely administered except for grade 3 lung injury. The TCR-T cell infusion exhibited significant tumor response and early-onset CRS in patients with tumors that express NY-ESO-1 at high levels. The differentiation properties of the manufactured T cells may be prognostic for TCR-T-related CRS.Trial registration numberNCT02366546.

The nanoparticle complex of cholesteryl pullulan (CHP) and NY-ESO-1 antigen protein (CHP-NY-ESO-1) presents multiple epitope peptides to MHC class I and II pathways, leading to CD8+ and CD4+ T cell responses. Poly-ICLC is a synthetic, double-stranded RNA, an agonist of toll-like receptor (TLR)-3, and a cytoplasmic receptor of melanoma differentiation-associated gene (MDA)-5. It should be a suitable immune adjuvant of cancer vaccine to overcome the inhibitory tumor microenvironment. We conducted a phase 1 clinical trial of CHP-NY-ESO-1 with poly-ICLC in patients with advanced or recurrent esophageal cancer. CHP-NY-ESO-1/poly-ICLC (μg/mg) was administered at a dose of 200/0.5 or 200/1.0 (cohorts 1 and 2, respectively) every 2 weeks for a total of six doses. The primary endpoints were safety and immune response. The secondary endpoint was tumor response. In total, 16 patients were enrolled, and six patients in each cohort completed the trial. The most common adverse event (AE) was injection site skin reaction (86.7%). No grade 3 or higher drug-related AEs were observed. No tumor responses were observed, and three patients (30%) had stable disease. The immune response was comparable between the two cohorts, and all patients (100%) achieved antibody responses with a median of 2.5 vaccinations. Comparing CHP-NY-ESO-1 alone to the poly-ICLC combination, all patients in both groups exhibited antibody responses, but the titers were higher in the combination group. In a mouse model, adding anti-PD-1 antibody to the combination of CHP-NY-ESO-1/poly-ICLC suppressed the growth of NY-ESO-1-expressing tumors. Combining the vaccine with PD-1 blockade holds promise in human trials.

The development of a new large-scale purification protocol is required for research on the reliable bioactivity and drug discovery of extracellular vesicles (EVs). To address this issue, herein, we propose an effective method for preparing high-performance exosomes (EXOs) by using an anion-exchange method. Cytotoxic T-lymphocyte (CTL) EVs from 4 L of culture supernatant through a 220 nm cut-off filter are divided into two populations at a deproteinization rate of over 99.97%, which are eluted at low (0.15 M-0.3 M) and high (0.3 M-0.5 M) NaCl concentrations (approximately 2 × 10

Cholesteryl pullulan (CHP) is a novel antigen delivery system. CHP and New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen complexes (CHP-NY-ESO-1) present multiple epitope peptides to the MHC class I and II pathways. Adjuvants are essential for cancer vaccines. MIS416 is a non-toxic microparticle that activates immunity via the nucleotide-binding oligomerization domain 2 (NOD2) and TLR9 pathways. However, no reports have explored MIS416 as a cancer vaccine adjuvant. We conducted a first-in-human clinical trial of CHP-NY-ESO-1 with MIS416 in patients with NY-ESO-1-expressing refractory solid tumors. CHP-NY-ESO-1/MIS416 (μg/μg) was administered at 100/200, 200/200, 200/400 or 200/600 (cohorts 1, 2, 3 and 4, respectively) every 2 weeks for a total of 6 doses (treatment phase) followed by one vaccination every 4 weeks until disease progression or unacceptable toxicity (maintenance phase). The primary endpoints were safety and tolerability, and the secondary endpoint was the immune response. In total, 26 patients were enrolled. Seven patients (38%) continued vaccination in the maintenance phase. Grade 3 drug-related adverse events (AEs) were observed in six patients (23%): anorexia and hypertension were observed in one and five patients, respectively. No grade 4–5 drug-related AEs were observed. Eight patients (31%) had stable disease (SD). Neither augmentation of the NY-ESO-1-specific IFN-γ-secreting CD8+ T cell response nor an increase in the level of anti-NY-ESO-1 IgG1 was observed as the dose of MIS416 was increased. In a preclinical study, adding anti-PD-1 monoclonal antibody to CHP-NY-ESO-1 and MIS416 induced significant tumor suppression. This combination therapy is a promising next step. Electronic supplementary material The online version of this article (10.1007/s00262-020-02483-1) contains supplementary material, which is available to authorized users.

Extracellular vesicles (EVs) are released by all types of mammalian cells for cell-cell communication. In this study, surface glycans on EVs are compared in terms of their cell type, size, and isolation method to examine whether EV glycan profiles by lectin microarray can be used to define EV subpopulations. Moreover, EVs are glycoengineered with four distinctive surface glycan patterns and evaluated their cellular uptake efficiencies for potential drug delivery applications. Both similarities and differences in glycan patterns are identified on EVs obtained under each experimental condition. EV size- and isolation method-dependent lectin-binding patterns are observed. Moreover, cellular uptake behaviors of EVs are affected by EV glycan profiles and acceptor cells. The in vivo biodistribution of EVs is also dependent on their glycan profile. These results suggest that EV surface glycans are a potential novel indicator of EV heterogeneity, and glycoengineering is a useful approach to regulate cell-EV interactions for biomedical applications.

IntroductionAdoptive cell transfer of genetically engineered T cells is a promising treatment for malignancies; however, there are few ideal cancer antigens expressed on the cell surface, and the development of chimeric antigen receptor T cells (CAR-T cells) for solid tumour treatment has been slow. CAR-T cells, which recognise major histocompatibility complex and peptide complexes presented on the cell surface, can be used to target not only cell surface antigens but also intracellular antigens. We have developed a CAR-T-cell product that recognises the complex of HLA-A*02:01 and an epitope of the MAGE-A4 antigen equipped with a novel signalling domain of human GITR (investigational product code: MU-MA402C) based on preclinical studies.Methods and analysisThis is a dose-escalation, multi-institutional, phase 1 study to evaluate the tolerability and safety of MU-MA402C for patients with MAGE A4-positive and HLA-A*02:01-positive unresectable advanced or recurrent solid cancer. Two dose cohorts are planned: cohort 1, MU-MA402C 2×108/person; cohort 2, MU-MA402C 2×109/person. Prior to CAR-T-cell infusion, cyclophosphamide (CPA) and fludarabine (FLU) will be administered as preconditioning chemotherapy. Three evaluable subjects per cohort, for a total of 6 subjects (maximum of 12 subjects), will be recruited for this clinical trial. The primary endpoints are safety and tolerability. The severity of each adverse event will be evaluated in accordance with Common Terminology Criteria for Adverse Events V.5.0. The secondary endpoint is efficacy. Antitumour response will be evaluated according to Response Evaluation Criteria in Solid Tumours V.1.1.Ethics and disseminationThis clinical trial will be conducted in accordance with the current version of Good Clinical Practice. The protocol was approved by the Clinical Research Ethics Review Committee of Mie University Hospital (approval number F-2021-017). The trial results will be published in peer-reviewed journals and/or disseminated through international conferences.Trial registration numberjRCT2043210077.

The anti-tumor efficacy of TCR-engineered T cells in vivo depends largely on less-differentiated subsets such as T cells with naive-like T cell (TN) phenotypes with greater expansion and long-term persistence. To increase these subsets, we compared the generation of New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T cells under supplementation with either IL-2 or IL-7/IL-15. PBMCs were transduced with MS3II-NY-ESO-1-siTCR retroviral vector. T cell generation was adapted from a CD19-specific CART cell production protocol. Comparable results in viability, expansion and transduction efficiency of T cells under stimulation with either IL-2 or IL-7/IL-15 were observed. IL-7/IL-15 led to an increase of CD4+ T cells and a decrease of CD8+ T cells, enriched the amount of TN among CD4+ T cells but not among CD8+ T cells. In a 51Cr release assay, similar specific lysis of NY-ESO-1-positive SW982 sarcoma cells was achieved. However, intracellular cytokine staining revealed a significantly increased production of IFN-γ and TNF-α in T cells generated by IL-2 stimulation. To validate these unexpected findings, NY-ESO-1-specific T cell production was evaluated in another protocol originally established for TCR-engineered T cells. IL-7/IL-15 increased the proportion of TN. However, the absolute number of TN did not increase due to a significantly slower expansion of T cells with IL-7/IL-15. In conclusion, IL-7/IL-15 does not seem to be superior to IL-2 for the generation of NY-ESO-1-specific T cells. This is in sharp contrast to the observations in CD19-specific CART cells. Changes of cytokine cocktails should be carefully evaluated for individual vector systems.

Background Cancer testis (CT) antigens are promising targets for cancer immunotherapies such as cancer vaccines and genetically modified adoptive T cell therapy. In this study, we evaluated the expression of three CT antigens, melanoma-associated antigen A4 (MAGE-A4), New York oesophageal squamous cell carcinoma 1 (NY-ESO-1) and sarcoma antigen gene (SAGE). Methods MAGE-A4, NY-ESO-1 and/or SAGE antigen expression in tumour samples was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Informed consent was obtained from individuals prior to study enrolment. Results In total, 585 samples in 21 tumour types were evaluated between June 2009 and March 2018. The positive expression rates of these CT antigens were as follows: MAGE-A4, 34.6% (range, 30.7–38.7); NY-ESO-1, 21.0% (range, 17.2–25.1); and SAGE, 21.8% (range, 18.5–25.4). The MAGE-A4 antigen was expressed in 54.9% of oesophageal cancers, 37.5% of head and neck cancers, 35.0% of gastric cancers and 34.2% of ovarian cancers; the NY-ESO-1 antigen was expressed in 28.6% of lung cancers, 25.3% of oesophageal cancers and 22.6% of ovarian cancers; and the SAGE antigen was expressed in 35.3% of prostate cancers, 32.9% of oesophageal cancers and 26.3% of ovarian cancers. The most common tumour type in this study was oesophageal cancer. MAGE-A4, NY-ESO-1 and SAGE antigen expression were assessed in 214 oesophageal cancer samples, among which 24 (11.2%) were triple-positive, 58 (27.1%) were positive for any two, 59 (27.6%) were positive for any one, and 73 (34.1%) were triple negative. Conclusions Oesophageal cancer exhibited a relatively high rate of CT antigen mRNA expression positivity.

Exosomes are representative extracellular vesicles (EV) derived from multivesicular endosomes (MVE) and have been described as new particles in the communication of neighborhood and/or distant cells by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and nucleotides including micro (mi) RNAs. Exosomes from immune cells and tumor cells act in part as a regulator in tumor immunology. CD8+ T cells that show potent cytotoxic activity against tumor cells reside as an inactive naive form in the T-cell zone of secondary lymphoid organs. Once receiving tumor-specific antigenic stimulation by dendritic cells (DC), CD8+ T cells are activated and differentiated into effector CTL. Subsequently, CTL circulate systemically, infiltrate into tumor lesions through the stromal neovasculature where mesenchymal stromal cells, for example, mesenchymal stem cells (MSC) and cancer-associated fibroblasts (CAF), abundantly exist, destroy mesenchymal tumor stroma in an exosome-mediated way, go into tumor parenchyma, and attack tumor cells by specific interaction. DC-derived and regulatory T (Treg) cell-derived exosomes, respectively, promote and inhibit CTL generation in this setting. In this review, we describe the roles of exosomes from immune cells and tumor cells on the regulation of tumor progression.

Wilms' tumor 1 (WT1) is constantly expressed in leukemic cells of acute leukemia and myelodysplastic syndrome (MDS). A T-cell receptor (TCR) that specifically reacts with WT1 peptide in the context of HLA-A*24:02 has been identified. We conducted a first-in-human trial of TCR-gene transduced T-cell (TCR-T-cell) transfer in patients with refractory acute myeloblastic leukemia (AML) and high-risk MDS to investigate the safety and cell kinetics of the T cells. The WT1-specific TCR-gene was transduced to T cells using a retroviral vector encoding small interfering RNAs for endogenous TCR genes. The T cells were transferred twice with a 4-week interval in a dose-escalating design. After the second transfer, sequential WT1 peptide vaccines were given. Eight patients, divided into 2 dose cohorts, received cell transfer. No adverse events of normal tissue were seen. The TCR-T cells were detected in peripheral blood for 8 weeks at levels proportional to the dose administered, and in 5 patients, they persisted throughout the study period. The persisting cells maintained ex vivo peptide-specific immune reactivity. Two patients showed transient decreases in blast counts in bone marrow, which was associated with recovery of hematopoiesis. Four of 5 patients who had persistent T cells at the end of the study survived more than 12 months. These results suggest WT1-specific TCR-T cells manipulated by ex vivo culture of polyclonal peripheral lymphocytes survived in vivo and retained the capacity to mount an immune reaction to WT1. This trial was registered at www.umin.ac.jp as #UMIN000011519.

// Daisuke Muraoka 1,2 , Naohiro Seo 1 , Tae Hayashi 1 , Chisaki Hyuga-Amaike 1 , Kana Okamori 1 , Isao Tawara 3 , Naozumi Harada 1 and Hiroshi Shiku 1 1 Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan 2 Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan 3 Department of Hematology and Oncology, Mie University Graduate School of Medicine, Mie, Japan Correspondence to: Daisuke Muraoka, email: // Hiroshi Shiku, email: // Keywords : memory T cell, CTL function, STAP2, STAT signaling, SOCS3, Immunology and Microbiology Section, Immune response, Immunity Received : April 18, 2016 Accepted : February 01, 2017 Published : February 16, 2017 Abstract Long-surviving memory CD8 + T cells generated by stimulation with appropriate tumor-associated antigens are the most aggressive and persistent tumoricidal effectors. In this event of memory CD8 + T cell development, the signal transducer and activator of transcription (STAT) proteins function as the crucial intracellular signaling molecules, but the regulatory mechanism of STATs in CD8 + T cells is not fully understood. In this study, we report for the first time, by using murine vaccination models, that signal-transducing adaptor protein-2 (STAP2) maintains the cytotoxicity of long-lived memory CD8 + T cells by controlling a STAT3/suppressor of cytokine signaling 3 (SOCS3) cascade. Following T cell activation, STAP2 expression was transiently reduced but was subsequently recovered and augmented. Analysis using small-interfering RNA (siRNA) demonstrated that restored STAP2 expression was associated with the activation of STAT3/SOCS3 signals and maintenance of cytotoxic T lymphocytes (CTLs) secondary responses by preventing their differentiation into terminal effector cells. Notably, this STAP2-dependent memory differentiation was observed in the spleen, but not in the lymph nodes (LNs). These findings indicate an essential role for STAP2 in the generation of a high-quality memory CD8 + CTLs periphery, and suggest the therapeutic potential of STAP2 in cancer patients.

Proteomic analysis requires protein tags that enable high-throughput handling; however, versatile tags that can be used in in vitro expression systems are currently lacking. In this study, we developed an insoluble protein tag, INSOL-tag, derived from human transcription factor MafG. The INSOL-tagged target protein is expressed in a eukaryotic in vitro expression system and recovered as a pellet following centrifugation at 19,000 × g for 20 min. Comparisons of the target protein recovery rates of GST-tag and INSOL-tag using 111 cytoplasmic proteins revealed a fourfold increase in the yield of INSOL-tagged proteins. Using 267 cancer antigens purified with INSOL-tag, we subsequently developed an INSOL-CTA array method, for profiling autoantibodies in sera of cancer patients. The detection limit of the array was approximately 11.1 pg IgG, and the correlation with ELISA was high (R2 = .993, .955). Moreover, when autoantibody profiling of digestive cancer patient sera was performed, antigen spreading was observed. These data suggest that INSOL-tag is a versatile tag that can insolubilize a wide range of target proteins. It is therefore expected to become a powerful tool in comprehensive protein preparation for protein arrays, antibody production, and mass spectrometry.

Adoptive T cell therapy using tumor-specific T cells or TCR-modified T cells is a promising next-generation immunotherapy. The major source of tumor-reactive T cells is PD-1+ tumor-infiltrating lymphocytes (TILs). In contrast, PD-1- TILs have received little attention. Here, we analyzed the TCR-β repertoires of PD-1- and PD-1+ CD8+ TILs derived from colorectal cancer and breast cancer. Approximately 40-60% of the PD-1+ population consisted of oligoclonal populations in both colorectal cancer and breast cancer. In contrast, approximately 37% of the PD-1- population consisted of an oligoclonal population in colorectal cancer, whereas 14% of them were oligoclonal in breast cancer. In colorectal cancer, the TCR repertoires of PD-1- CD8+ TILs and PD-1+ CD8+ TILs hardly overlapped. Interestingly, clonally expanded CD8+ TILs in primary tumors and the metastases expressing the same clonotypic TCR showed the same phenotype regarding the PD-1-expression. These results suggest that the intrinsic properties of TCRs determine the fate of TILs in terms of whether they become PD-1+ or PD-1- in the tumor microenvironment. Further functional analysis of TCRs in TILs will allow us to better understand the regulatory mechanisms for PD-1 expression on TILs and may contribute to tumor immunotherapy.

NY-ESO-1-specific T cells have shown promising activity in the treatment of soft tissue sarcoma (STS). However, standardized protocols for their generation are limited. Particularly, cost-effectiveness considerations of cell production protocols are of importance for conducting clinical studies. In this study, two different NY-ESO-1-specific T cell production protocols were compared. Major differences between protocols 1 and 2 include culture medium, interleukin-2 and retronectin concentrations, T cell activation strategy, and the transduction process. NY-ESO-1-specific T cells generated according to the two protocols were investigated for differences in cell viability, transduction efficiency, T cell expansion, immunophenotype as well as functionality. NY-ESO-1-specific T cells showed similar viability and transduction efficiency between both protocols. Protocol 1 generated higher absolute numbers of NY-ESO-1-specific T cells. However, there was no difference in absolute numbers of NY-ESO-1-specific T cell subsets with less-differentiated phenotypes accounting for efficient in vivo expansion and engraftment. Furthermore, cells generated according to protocol 1 displayed higher capacity of TNF-&alpha, generation, but lower cytotoxic capacities. Overall, both protocols provided functional NY-ESO-1-specific T cells. However, compared to protocol 1, protocol 2 is advantageous in terms of cost-effectiveness. Cell production protocols should be designed diligently to achieve a cost-effective cellular product for further clinical evaluation.

Purpose: Mogamulizumab (Mog), a humanized anti-CC chemokine receptor 4 (CCR4) mAb that mediates antibody-dependent cellular cytotoxicity (ADCC) using FcγR IIIa (CD16)-expressing effector cells, has recently been approved for treatment of CCR4-positive adult T-cell leukemia (ATL) in Japan. However, Mog failure has sometimes been observed in patients who have accompanying chemotherapy-associated lymphocytopenia. In this study, we examined whether adoptive transfer of artificial ADCC effector cells combined with Mog would overcome this drawback. Experimental Design: We lentivirally gene-modified peripheral blood T cells from healthy volunteers and ATL patients expressing the affinity-increased chimeric CD16-CD3ζ receptor (cCD16ζ-T cells). Subsequently, we examined the ADCC effect mediated by those cCD16ζ-T cells in the presence of Mog against ATL tumor cells both in vitro and in vivo. Results: cCD16ζ-T cells derived from healthy donors killed in vitro Mog-opsonized ATL cell line cells (n = 7) and primary ATL cells (n = 4) depending on both the number of effector cells and the dose of the antibody. cCD16ζ-T cells generated from ATL patients (n = 3) also exerted cytocidal activity in vitro against Mog-opsonized autologous ATL cells. Using both intravenously disseminated model (n = 5) and subcutaneously inoculated model (n = 4), coadministration of Mog and human cCD16ζ-T cells successfully suppressed tumor growth in xenografted immunodeficient mice, and significantly prolonged their survival (P < 0.01 and P = 0.02, respectively). Conclusions: These data strongly suggest clinical feasibility of the novel combined adoptive immunotherapy using cCD16ζ-T cells and Mog for treatment of aggressive ATL, particularly in patients who are ineligible for allogeneic hematopoietic stem cell transplantation. Clin Cancer Res; 22(17); 4405–16. ©2016 AACR.

Aim: To investigate the antigen spreading pattern in the CHP-MAGE-A4-vaccinated patients and analyze the clinical relevance of antigen spreading pattern as a surrogate marker of patient survival. Materials & methods: 12 patients who had been injected with 300 μg of CHP-MAGE-A4 and 0.5 Klinische Einheit of OK-432 in more than five vaccinations were analyzed. Results: Increases in the anti-MAGE-A4-specific antibody response were observed in eight patients (66.7%), compared with six patients (50%) for anti-NY-ESO-1 and five patients (41.7%) for anti-MAGE-A3 after five vaccinations. We identified frequent antigen spreading following MAGE-A4 vaccinations without associations with the clinical response or patient prognosis. Conclusion: Antigen spreading pattern might reflect tumor shrinkage as a response to treatment and treatment history (clinical trial registration number: UMIN000001999).

Melanoma antigen family A4 (MAGEA4), a cancer/testis antigen, is overexpressed and is thus an immunotherapy target in various malignant tumors, including non-small cell lung cancer. However, whether MAGEA4 induces or inhibits the apoptosis of lung cancer cells remains controversial, as is its prognostic significance, particularly since there is no reliable method with which to detect MAGEA4 specifically. In this study, we optimized assay conditions to detect MAGEA4 based on cells transiently transfected with MAGEA genes, and found that MAGEA4 was expressed in four of eight non-small cell lung cancer cell lines, and in 25.4% of clinical lung cancer specimens. We also found that MAGEA4 overexpression decreased apoptosis, as measured by the levels of cleaved caspase-3 in stably transfected 293F cells. Notably, patients with nuclear MAGEA4, but not p53 expression exhibited a significantly poorer survival than those expressing both nuclear MAGEA4 and p53. Indeed, multivariate analysis identified nuclear MAGEA4 as an independent prognostic factor (P=0.0042), albeit only in the absence of p53. In this study, to the best of our knowledge, we are the first to demonstrate that the function and prognostic value of MAGEA4 depends on its subcellular localization and on the p53 status.

The recent success of chimeric antigen receptor (CAR)-T cell therapy for treatment of hematologic malignancies supports further development of treatments for both liquid and solid tumors. However, expansion of CAR-T cell therapy is limited by the availability of surface antigens specific for the tumor while sparing normal cells. There is a rich diversity of tumor antigens from intracellularly expressed proteins that current and conventional CAR-T cells are unable to target. Furthermore, adoptively transferred T cells often suffer from exhaustion and insufficient expansion, in part, because of the immunosuppressive mechanisms operating in tumor-bearing hosts. Therefore, it is necessary to develop means to further activate and expand those CAR-T cells in vivo. The Wilms tumor 1 (WT1) is an intracellular oncogenic transcription factor that is an attractive target for cancer immunotherapy because of its overexpression in a wide range of leukemias and solid tumors, and a low level of expression in normal adult tissues. In the present study, we developed CAR-T cells consisting of a single chain variable fragment (scFv) specific to the WT1235-243/HLA-A*2402 complex. The therapeutic efficacy of our CAR-T cells was demonstrated in a xenograft model, which was further enhanced by vaccination with dendritic cells (DCs) loaded with the corresponding antigen. This enhanced efficacy was mediated, at least partly, by the expansion and activation of CAR-T cells. CAR-T cells shown in the present study not only demonstrate the potential to expand the range of targets available to CAR-T cells, but also provide a proof of concept that efficacy of CAR-T cells targeting peptide/major histocompatibility complex can be boosted by vaccination.

The central tumoricidal activity of anticancer monoclonal antibodies (mAb) is exerted by FcγR IIIa (CD16)–expressing effector cells in vivo via antibody-dependent cell-mediated cytotoxicity (ADCC), as observed for natural killer (NK) cells. In practice, chemotherapy-induced leukopenia and exhaustion of NK cells resulting from ADCC often hamper the clinical efficacy of cancer treatment. To circumvent this drawback, we examined in vivo the feasibility of T cells, gene-modified to express a newly generated affinity-matured (158V/V) chimeric CD16-CD3ζ receptor (cCD16ζ-T cells), as a transferable alternative effector for cancer mAb therapy. cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human interleukin-2 (rhIL-2), and they successfully displayed ADCC-mediated tumoricidal activity in vitro. During ADCC, ligation of opsonized cancer cells to the introduced cCD16ζ-T cells stimulated the effector cells to produce proinflammatory cytokines and release toxic granules through the activation of the Nuclear factor of activated T cells (NFAT) pathway after phosphorylation of the CD3ζ chain. In parallel, these stimulated cCD16ζ-T cells transiently proliferated and differentiated into effector memory T cells. In contrast, NK cells activated by rhIL-2 displayed similar ADCC activity, but failed to proliferate. Human cCD16ζ-T cells infused concomitantly with anti-CD20 mAb synergistically inhibited the growth of disseminated Raji cells, a CD20+ lymphoma cell line, in immunodeficient mice, whereas similarly infused rhIL-2–treated NK cells survived for a shorter time and displayed less effective tumor suppression. Our findings strongly suggest the clinical feasibility of cCD16ζ-T cells as adoptively transferable ADCC effector cells that could potentially enhance the clinical responses mediated by currently available anticancer mAbs. Cancer Immunol Res; 2(3); 249–62. ©2014 AACR.

Although adult T-cell leukemia (ATL) has a poor prognosis, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) in some cases suggests that a cellular immune-mediated strategy can be effective. So far, however, no effective target for anti-ATL immunotherapy has been defined. Here we demonstrated for the first time that human telomerase reverse transcriptase (hTERT) is a promising therapeutic target for ATL, and we developed a novel redirected T-cell-based immunotherapy targeting hTERT. hTERT messenger RNA was produced abundantly in ATL tumor cells but not in steady-state normal cells. Rearranged human leukocyte antigen-A*24:02 (HLA-A*24:02) -restricted and hTERT461-469 nonameric peptide-specific T-cell receptor (TCR) α/β genes were cloned from our previously established cytotoxic T lymphocyte clone (K3-1) and inserted into a novel retroviral TCR expression vector encoding small interfering RNAs for endogenous TCR genes in redirected T cells (hTERT-siTCR vector). Consequently, allogeneic or autologous gene-modified CD8(+) T cells prepared using the hTERT-siTCR vector successfully killed ATL tumor cells, but not normal cells including steady-state hematopoietic progenitors, in an HLA-A*24:02-restricted manner both in vitro and in vivo. Our experimental observations support the development of a novel hTERT-targeting redirected T-cell-based adoptive immunotherapy for ATL patients, especially those for whom suitable allo-HSCT donors are lacking.

Forkhead box protein p3 (Foxp3) is crucial to the development and suppressor function of regulatory T cells (Tregs) that have a significant role in tumor-associated immune suppression. Development of small molecule inhibitors of Foxp3 function is therefore considered a promising strategy to enhance anti-tumor immunity. In this study, we developed a novel cell-based assay system in which the NF-κB luciferase reporter signal is suppressed by the co-expressed Foxp3 protein. Using this system, we screened our chemical library consisting of approximately 2,100 compounds and discovered that a cancer chemotherapeutic drug epirubicin restored the Foxp3-inhibited NF-κB activity in a concentration-dependent manner without influencing cell viability. Using immunoprecipitation assay in a Treg-like cell line Karpas-299, we found that epirubicin inhibited the interaction between Foxp3 and p65. In addition, epirubicin inhibited the suppressor function of murine Tregs and thereby improved effector T cell stimulation in vitro. Administration of low dose epirubicin into tumor-bearing mice modulated the function of immune cells at the tumor site and promoted their IFN-γ production without direct cytotoxicity. In summary, we identified the novel action of epirubicin as a Foxp3 inhibitor using a newly established luciferase-based cellular screen. Our work also demonstrated our screen system is useful in accelerating discovery of Foxp3 inhibitors.

Exosomes are a valuable biomaterial for the development of novel nanocarriers as functionally advanced drug delivery systems. To control and modify the performance of exosomal nanocarriers, we developed hybrid exosomes by fusing their membranes with liposomes using the freeze–thaw method. Exosomes embedded with a specific membrane protein isolated from genetically modified cells were fused with various liposomes, confirming that membrane engineering methods can be combined with genetic modification techniques. Cellular uptake studies performed using the hybrid exosomes revealed that the interactions between the developed exosomes and cells could be modified by changing the lipid composition or the properties of the exogenous lipids. These results suggest that the membrane-engineering approach reported here offers a new strategy for developing rationally designed exosomes as hybrid nanocarriers for use in advanced drug delivery systems.

Adoptive cell therapy using tumor-specific T cells is a promising strategy for treating patients with malignancy. However, accumulating evidences have demonstrated that optimal function of tumor-reactive T cells is often attenuated by negative regulatory signal(s) delivered through receptors, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1), and their cognate ligands. Although systemic blocking of these molecules needs careful attention on the risk of uncontrolled immune activation, selective inhibition of negative signals in tumor-specific T cells by their genetic modification is an attractive approach to overcome immunological suppression in cancer patients. Here, we demonstrate the improved effector functions of tumor-specific CD4(+) and CD8(+) human T cells by small interfering RNA (siRNA) -mediated silencing of PD-1 ligands, PD-L1 or PD-L2. Tumor antigen MAGE-A4-specific human T-cell clones upregulated the expression of PD-1 ligands upon activation. siRNA-mediated knockdown of PD-L1 or -L2 enhanced the interferon-γ production and antigen-specific cytotoxicity of these cells. Peripheral blood mononuclear cells transduced with a retroviral vector encoding MAGE-A4-specific T-cell receptor α/β chains also increased their effector functions by this modification. These results suggest that siRNA-mediated knockdown of PD-1 ligands is an attractive strategy to inhibit a negative regulatory mechanism of tumor-specific T cells resulting in enhanced efficacy of adoptive T-cell therapy of cancer using genetically modified autologous lymphocytes.

Adoptive cell therapy with lymphocytes that have been genetically engineered to express tumor-reactive T-cell receptors (TCR) is a promising approach for cancer immunotherapy. We have been exploring the development of TCR gene therapy targeting cancer/testis antigens, including melanoma-associated antigen (MAGE) family antigens, that are ideal targets for adoptive T-cell therapy. The efficacy of TCR gene therapy targeting MAGE family antigens, however, has not yet been evaluated in vivo. Here, we demonstrate the in vivo antitumor activity in immunodeficient non-obese diabetic/SCID/γc(null) (NOG) mice of human lymphocytes genetically engineered to express TCR specific for the MAGE-A4 antigen. Polyclonal T cells derived from human peripheral blood mononuclear cells were transduced with the αβ TCR genes specific for MAGE-A4, then adoptively transferred into NOG mice inoculated with MAGE-A4 expressing human tumor cell lines. The transferred T cells maintained their effector function in vivo, infiltrated into tumors, and inhibited tumor growth in an antigen-specific manner. The combination of adoptive cell therapy with antigen peptide vaccination enhanced antitumor activity, with improved multifunctionality of the transferred cells. These data suggest that TCR gene therapy with MAGE-A4-specific TCR is a promising strategy to treat patients with MAGE-A4-expressing tumors; in addition, the acquisition of multifunctionality in vivo is an important factor to predict the quality of the T-cell response during adoptive therapy with human lymphocytes.

Wilms' tumor gene (WT1), which is expressed in human pancreatic cancer (PC), is a unique tumor antigen recognized by T-cell-mediated antitumor immune response. Gemcitabine (GEM), a standard therapeutic drug for PC, was examined for the regulation of WT1 expression and the sensitizing effect on PC cells with WT1-specific antitumor immune response. Expression of WT1 was examined by quantitative PCR, immunoblot analysis, and confocal microscopy. Antigenic peptide of WT1 presented on HLA class I molecules was detected by mass spectrometry. WT1-specific T-cell receptor gene-transduced human T cells were used as effecter T cells for the analysis of cytotoxic activity. GEM treatment of human MIAPaCa2 PC cells enhanced WT1 mRNA levels, and this increase is associated with nuclear factor kappa B activation. Tumor tissue from GEM-treated MIAPaCa2-bearing SCID mice also showed an increase in WT1 mRNA. Some human PC cell lines other than MIAPaCa2 showed up-regulation of WT1 mRNA levels following GEM treatment. GEM treatment shifted WT1 protein from the nucleus to the cytoplasm, which may promote proteasomal processing of WT1 protein and generation of antigenic peptide. In fact, presentation of HLA-A*2402-restricted antigenic peptide of WT1 (CMTWNQMNL) increased in GEM-treated MIAPaCa2 cells relative to untreated cells. WT1-specific cytotoxic T cells killed MIAPaCa2 cells treated with an optimal dose of GEM more efficiently than untreated MIAPaCa2 cells. GEM enhanced WT1 expression in human PC cells and sensitized PC cells with WT1-specific T-cell-mediated antitumor immune response.

Deletion of the long arm of chromosome 20 (del 20q) has been observed in patients with myelodysplastic syndrome (MDS) or myeloid malignancies. We experienced an MDS female case of del 20q accompanied by clusters of plasmacytic cells in bone marrow. Her bone marrow cells showed morphological abnormalities in three lineages and the chromosomal abnormality of 46, XX, del (20) (q11.2q13.3). Although the percentage of plasma cells was low in free cells, such cells showed nuclear abnormalities. In bone marrow clots, we also observed clusters of anti-CD38 and anti-CD138 antibody-positive cells. According to the FAB or WHO classification, the diagnosis was unclear. Therefore, we were obliged to term this case as MDS with plasma cell dysplasia. This patient was considered to be a rare case of MDS related to abnormalities in myeloid and B-lymphoid cells.

Combination vaccines of the NY-ESO-1 protein complexed with cholesteryl pullulan (CHP), CHP-NY-ESO-1, and the truncated 146HER2 protein with CHP, CHP-HER2, were subcutaneously administered with the immuno-adjuvant OK-432 to eight esophageal cancer patients. Vaccination was well-tolerated. NY-ESO-1- and HER2-specific antibody responses were analyzed using the patients' sera and samples from previous single CHP-NY-ESO-1 or CHP-HER2 vaccine trial. The responses to NY-ESO-1 in the combination vaccine study were comparable to the single vaccine. For responses to HER2, there were fewer antibody responses in the combination vaccines. Although there were marked individual variations in the antibody responses to the NY-ESO-1 and HER2 antigens, the reaction patterns to these antigens were comparable within each patient. Antibodies to OK-432 were not augmented. Protein cancer vaccines targeting multiple antigens are feasible.

The gene expression profile of peripheral gammadelta T-cell lymphoma (gammadeltaTCL) has not been investigated. Using oligonucleotide microarrays, we analyzed total RNA from 7 patients with gammadeltaTCL (4 hepatosplenic, 1 cutaneous, 1 intestinal, and 1 thyroidal) and 27 patients with alphabetaTCL (11 peripheral TCL-unspecified, 15 angioimmunoblastic TCL, and 1 hepatosplenic). Unsupervised microarray analyses classified all hepatosplenic gammadeltaTCLs into a single cluster, whereas other gammadeltaTCLs were scattered within the alphabetaTCL distribution. We identified a T-cell receptor signature gene set, which accurately classified gammadeltaTCL and alphabetaTCL. A classifier based on gene expression under supervised analysis correctly identified gammadeltaTCL. One case of hepatosplenic alphabetaTCL was placed in the gammadeltaTCL grouping. gammadeltaTCL signature genes included genes encoding killer cell immunoglobulin-like receptors and killer cell lectin-like receptors. Our results indicate that hepatosplenic gammadeltaTCL is a distinct form of peripheral TCL and suggest that nonhepatosplenic gammadeltaTCLs are heterogeneous in gene expression.

NY-ESO-1 antigen is a prototype of a class of cancer/testis antigens. We carried out a clinical trial using NY-ESO-1 whole protein as a cancer vaccine for 13 advanced cancer patients. We have recently reported that vaccine elicited humoral and cellular immune responses in 9 cancer patients including 4 esophageal cancer patients, and clinical responses were also observed in 4 of 5 evaluable patients. In this study, we analyzed the responses in 8 esophageal cancer patients including 4 newly enrolled patients. Patients were injected subcutaneously at biweekly intervals with NY-ESO-1 recombinant protein formulated with cholesterol-bearing hydrophobized pullulan. Induction of antibody, and CD4 and CD8 T-cell responses were observed in 7, 7 and 6 patients, respectively, out of 8 patients. 1 PR, 2 SD and 2 mixed clinical responses were observed in 6 evaluable patients. No significant adverse events were observed. Furthermore, we analyzed NY-ESO-1 and MHC class I expression and the infiltration of immune cells into tumor samples obtained before and after vaccination from 4 patients by immunohistochemistry. The results showed 2 patients with disappearance of CD4 and CD8 T-cell infiltration, 1 patient with increase in the number of CD68(+) macrophages and 1 patient with tumor antigen loss in the progressive tumors following vaccinations. The induction of NY-ESO-1 immunity and some preferable clinical outcomes were observed in esophageal cancer patients by vaccination with NY-ESO-1. However, the tumors grew eventually by various mechanisms after vaccination.

Background De novo CD5-positive diffuse large B-cell lymphoma (CD5+ DLBCL) is clinicopathologically and genetically distinct from CD5-negative (CD5−) DLBCL and mantle cell lymphoma. The aim of this retrospective study was to clarify the histopathological spectrum and obtain new information on the therapeutic implications of CD5+ DLBCL. Design and Method From 1984 to 2002, 120 patients with CD5+ DLBCL were selected from 13 collaborating institutes. We analyzed the relationship between their morphological features and long-term survival. The current series includes 101 patients described in our previous study. Results Four morphological variants were identified: common (monomorphic) (n=91), giant cell-rich (n=13), polymorphic (n=14), and immunoblastic (n=2). Intravascular or sinusoidal infiltration was seen in 38% of the cases. BCL2 protein expression in CD5+ DLBCL was more frequent than in CD5− DLBCL ( p =0.0003). Immunohistochemical analysis in 44 consecutive cases of CD5+ DLBCL revealed that 82% of these cases (36/44) were non-germinal center B-cell type DLBCL. The 5-year overall survival rate of the patients with CD5+ DLBCL was 38% after a median observation time of 81 months. Patients with the common variant showed a better prognosis than those with the other three variants ( p =0.011), and this was confirmed on multivariate analysis. Overall, 16 patients (13%) developed central nervous system recurrence. Conclusions Our study revealed the morphological spectrum of CD5+ DLBCL, found that the incidence of central nervous system recurrence in this form of lymphoma in high, confirmed that CD5+ DLBCL frequently expresses BCL2 protein and showed that it is mainly included in the non-germinal center B-cell type of DLBCL.

The CHP-HER2 vaccine, comprising truncated 146HER2 protein complexed with nanogels of cholesteryl pullulan (CHP), is a novel protein antigen vaccine that elicits 146HER2-specific CD8(+) and CD4(+) T-cell immune responses in patients with HER2-expressing tumors. We analyzed the humoral responses in patients vaccinated with CHP-HER2 and those with CHP-HER2 plus granulocyte-macrophage colony-stimulating factor (GM-CSF). The vaccine was injected subcutaneously at a dose of 300 microg protein. Nine patients received the vaccine alone over the first four injections, followed by CHP-HER2 with GM-CSF or OK-432, whereas six received CHP-HER2 plus GM-CSF from the first cycle. 146HER2-specific IgG antibodies were induced in 14 patients, who were negative at baseline. The antibodies became detectable after the second or third vaccination and reached plateau levels after the third or fourth cycle in patients vaccinated with CHP-HER2 plus GM-CSF. In contrast, the antibodies appeared only after the third to sixth vaccination and the plateau appeared after the fourth to eighth cycle in patients vaccinated with the CHP-HER2 vaccine alone over the first four cycles. The antibodies induced by the vaccine were not reactive with HER2 antigen expressed on the cell surface in any of the patients. Epitope analysis using overlapping peptides revealed a single region in the 146HER2 protein, amino acids 127-146, in eight patients who were initially vaccinated with CHP-HER2 alone. Similarly, the same HER2 region was recognized dominantly in patients vaccinated with GM-CSF. Our results indicate that CHP-HER2 induced HER2-specific humoral responses in patients with HER2-expressing tumors and that GM-CSF seems to accelerate the responses.

NY-ESO-1 specific humoral responses are frequently observed in patients with various types of NY-ESO-1 antigen expressing tumors. In a large proportion of NY-ESO-1 antibody-positive patients of NY-ESO-1-specific CD8 T-cells can also be detected suggesting that monitoring of the NY-ESO-1 specific humoral immune response may be a relevant and more practical surrogate for estimating the overall immune response against NY-ESO-1 in clinical vaccine studies. We have immunized 9 cancer patients with full length NY-ESO-1 protein formulated with cholesterol-bearing hydrophobized pullulan (CHP-NY-ESO-1) and investigated the humoral immune responses against NY-ESO-1. Seven patients were NY-ESO-1 antibody-negative and 2 patients were positive prior to vaccination. Vaccination with CHP-NY-ESO-1 resulted in the induction or increase of NY-ESO-1 antibody responses in all 9 patients immunized. Epitope analysis revealed 5 regions in the NY-ESO-1 protein molecule that were recognized by antibodies induced after vaccination. The 5 regions were also recognized by antibodies present in nonvaccinated, NY-ESO-1 antibody-positive cancer patients. A peptide spanning amino acids 91-108 was recognized in 6 out of 9 vaccinated patients and in 8 out of 9 nonvaccinated, sero-positive patients, being the most dominant antigenic epitope in NY-ESO-1 for antibody recognition in cancer patients. In conclusion, we showed that CHP-NY-ESO-1 protein vaccination had a potent activity for inducing humoral immune responses against NY-ESO-1 antigen in cancer patients. The antigenic epitopes recognized by antibodies in the vaccinated patients were similar to those recognized in cancer patients with spontaneous humoral immunity against NY-ESO-1.

The ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type I domain 13) activity was measured by a fluorescence resonance energy transfer (FRET) assay in the plasma of healthy volunteers and thrombotic thrombocytopenic purpura (TTP) patients to examine its usefulness in the diagnosis of TTP. The plasma levels of the ADAMTS13 activity did not show a normal distribution. Its median value was 107% (range: 55-170%) in healthy volunteers, but was significantly lower in patients with TTP (acquired or familial) and in patients with hematopoietic stem cell transplantation. However, it was not significantly lower in patients with antiphospholipid syndrome (APS). The ADAMTS13 activity by a FRET assay was closely correlated with that by the ADAMTS13 multimer method (r=0.816; p0.001). In 18 patients with less than 10% of ADAMTS13 activity by FRET assay, less than 10% of that by multimer assay was 16, thus suggesting a good correlation for a low level of ADAMTS13. These findings suggest that the ADAMTS13 FRET assay correlates well with the ADAMTS13 multimer method and it is therefore useful for making a diagnosis of TTP.

The development of cancer immunotherapies is progressing rapidly with a variety of technological approaches. They consist of "cancer vaccines", which are based on the idea of vaccination, "effector cell therapy", classified as passive immunotherapy, and "inhibition of immunosuppression", which intends to break immunological tolerance to autoantigens or immunosuppressive environments characterizing antitumor immune responses. Recent reports showing clinical evidence of efficacy of immune checkpoint inhibitors and adoptive immunotherapies with tumor-infiltrating lymphocytes and tumor-specific receptor gene-modified T cells indicate the beginning of a new era for cancer immunotherapy. This guidance summarizes ideas that will be helpful to those who plan to develop cancer immunotherapy. The aims of this guidance are to discuss and offer important points in early phase clinical studies of innovative cancer immunotherapy, with future progress in this field, and to contribute to the effective development of cancer immunotherapy aligned with the scope of regulatory science. This guidance covers cancer vaccines, effector cell therapy, and inhibition of immunosuppression, including immune checkpoint inhibitors.

To develop gene-modified T-cell-based antileukemia adoptive immunotherapy, concomitant administration of CD4(+) and CD8(+) T cells that have been gene modified using identical HLA class I-restricted leukemia antigen-specific T-cell receptor (TCR) gene transfer has not yet been fully investigated. Here, using CD4(+) and CD8(+) T cells that had been gene modified with a retroviral vector expressing HLA-A*24:02-restricted and Wilms' tumor 1 (WT1)-specific TCR-α/β genes and siRNAs for endogenous TCRs (WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells), we examined the utility of this strategy. WT1-siTCR/CD4(+) T cells sufficiently recognized leukemia cells in an HLA class I-restricted manner and provided target-specific Th1 help for WT1-siTCR/CD8(+) T cells. By using a xenografted mouse model, we found that WT1-siTCR/CD4(+) T cells migrated to leukemia sites and subsequently attracted WT1-siTCR/CD8(+) T cells via chemotaxis. Therapy-oriented experiments revealed effective enhancement of leukemia suppression mediated by concomitant administration of WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells. Importantly, this augmented efficacy in the presence of WT1-siTCR/CD4(+) T cells was correlated with longer survival and enhanced formation of memory T cells by WT1-siTCR/CD8(+) T cells. Collectively, our experimental findings strongly suggest that this strategy would be clinically advantageous for the treatment of human leukemia.

Tumor antigen-specific CD4+ T cells generally orchestrate and regulate immune cells to provide immune surveillance against malignancy. However, activation of antigen-specific CD4+ T cells is restricted at local tumor sites where antigen-presenting cells (APCs) are frequently dysfunctional, which can cause rapid exhaustion of anti-tumor immune responses. Herein, we characterize anti-tumor effects of a unique human CD4+ helper T-cell subset that directly recognizes the cytoplasmic tumor antigen, NY-ESO-1, presented by MHC class II on cancer cells. Upon direct recognition of cancer cells, tumor-recognizing CD4+ T cells (TR-CD4) potently induced IFN-γ-dependent growth arrest in cancer cells. In addition, direct recognition of cancer cells triggers TR-CD4 to provide help to NY-ESO-1-specific CD8+ T cells by enhancing cytotoxic activity and improving viability and proliferation in the absence of APCs. Notably, the TR-CD4 either alone or in collaboration with CD8+ T cells significantly inhibited tumor growth in vivo in a xenograft model. Finally, retroviral gene-engineering with T cell receptor (TCR) derived from TR-CD4 produced large numbers of functional TR-CD4. These observations provide mechanistic insights into the role of TR-CD4 in tumor immunity and suggest that approaches to utilize TR-CD4 will augment anti-tumor immune responses for durable therapeutic efficacy in cancer patients.

Purpose: We developed a complex of tumor antigen protein with a novel nanoparticle antigen delivery system of cholesteryl pullulan (CHP). To target HER2 antigen, we prepared truncated HER2 protein 1-146 (146HER2) complexed with CHP, the CHP-HER2 vaccine. We designed a clinical study to assess the safety of the vaccine and HER2-specific T-cell immune responses measured by the newly developed enzyme-linked immunospot assay with mRNA-transduced phytohemagglutinin-stimulated CD4+ T cells in HLA-A2402-positive patients with therapy-refractory HER2-expressing cancers.Experimental Design: Nine patients with various types of solid tumors were enrolled. Each patient was s.c. vaccinated biweekly with 300 μg of CHP-HER2 vaccine for three times followed by booster doses. HER2-specific T-cell responses were evaluated by enzyme-linked immunospot assay by targeting autologous phytohemagglutinin-stimulated CD4+ T cells transduced with 146HER2-encoding mRNA to cover both identified peptides and unknown epitopes for MHC class I and class II that might exist in the sequence of the vaccine protein.Results: CHP-HER2 vaccine was well tolerated; the only adverse effect was grade 1 transient skin reaction at the sites of vaccination. HER2-specific CD8+ and/or CD4+ T-cell immune responses were detected in five patients who received four to eight vaccinations, among whom both T-cell responses were detected in these patients. In four patients with CD8+ T-cell responses, two patients reacted to previously identified HER263-71 peptide and the other two reacted only to 146HER2 mRNA-transduced cells.Conclusions: CHP-HER2 vaccine was safe and induced HER2-specific CD8+ and/or CD4+ T-cell immune responses.

Summary Histone acetylation and deacetylation play fundamental roles in transcriptional regulation. We investigated the role of histone deacetylases (HDACs) in human adult haematopoiesis, using the structurally distinct HDAC inhibitors FK228 (depsipeptide) and Trichostatin A. When CD34 + cells were cultured with interleukin (IL)-3 or stem cell factor (SCF) + IL-3, FK228 (0AE5 ng/ml) specifically enhanced the generation of immature erythroid cells with a CD36 + glycophorin A (GPA) low phenotype. In semisolid cultures, FK228 promoted the formation of erythroid colonies by CD34 + cells with IL-3 and SCF + IL-3. Furthermore, upon exposure to FK228, CD34 + cell-derived CD36 + GPA ) cells were induced to form erythroid colonies with IL-3 alone. Conversely, FK228 inhibited the generation of CD36 + GPA high relatively mature erythroid cells from CD34 + cells in the presence of erythropoietin (EPO) and SCF + EPO. FK228 suppressed the EPO-mediated survival of CD36 + GPA low/- and CD36 + GPA high cells and induced their apoptosis. Similar effects were observed for trichostatin A in the generation of erythroid cells in IL-3- and EPO-containing cultures. These data suggest that HDACs negatively regulate the IL-3-mediated growth of early erythroid precursors by suppressing their responsiveness to IL-3, while playing an important role in EPO-mediated differentiation and survival of erythroid precursors. Our data revealed that HDACs have diverse functions in human adult erythropoiesis.

The progenitor cells of myelodysplastic syndrome (MDS) are thought to undergo a multistep process during their transformation into overt acute leukemia. In this study, the role of mutation of the KIT gene in the extracellular membrane, juxtamembrane and tyrosine kinase domains was investigated in 75 patients with MDS or MDS-derived leukemia (MDS-AML). Mutation was detected in 2 of 15 (13.3%) patients with refractory anemia with excess blasts transformation (RAEB-T), in 1 of 15 (6.6%) patients with chronic myelomonocytic leukemia (CMML), and in 5 of 26 (19.2%) patients with MDS-AML. However, no mutation was found in any of the nine patients with refractory anemia (RA) or the 10 patients with refractory anemia with excess blasts (RAEB). Of the mutations, five patients had changes at the same codon in tyrosine kinase domain, Asp816, while the remainder had unique mutations. These observations suggest that KIT gene mutations identified in the advanced stage of MDS, and genetic abnormality in the KIT gene, particularly at codon 816, might be additional events that contribute to the progression of MDS to AML.

We describe a patient with low-titer cold agglutinin disease (CAD) who developed mixed-type autoimmune hemolytic anemia (AIHA) and idiopathic thrombocytopenia following chicken pox infection. At least 1 year before admission to hospital, the patient had mild hemolytic anemia associated with low-titer cold agglutinins. A severe hemolytic crisis and thrombocytopenia (Evans' syndrome) occurred several days after infection with chicken pox, and the patient was referred to our hospital. Serological findings revealed the presence of both cold agglutinins and warm-reactive autoantibodies against erythrocytes, and the diagnosis was mixed-type AIHA. Following steroid therapy, the hemoglobin (Hb) level and platelet count improved. The patient was closely followed over a 10-year period with recurrent documented hemolysis after viral or bacterial infections. Warm-reactive autoantibodies have not been detected in the last 2 years, and only the immunoglobulin M anti-I cold agglutinins with a low titer and wide thermal amplitude have remained unchanged. Therefore, the patient has received at least 10 mg prednisolone daily to maintain a Hb level of 10 g/dL. To the best of our knowledge, no adult case of low-titer CAD that has evolved into mixed-type AIHA and Evans' syndrome after chicken pox infection has been previously reported in the literature.

Diffuse large B-cell lymphoma (DLBCL) accounts for 30% of non-Hodgkin's lymphomas and is known to comprise heterogeneous groups. We previously reported that CD5+ DLBCL is a clinically distinct subgroup of these tumors that is associated with poor prognosis. In our current study, we have used gene expression profiling technology in an attempt to identify new markers and to further characterize the biological features of CD5+ DLBCL. Candidate genes, which showed the greatest difference in expression between 22 CD5+ and 26 CD5− DLBCL cases, were selected from our screening and subjected to clustering analysis. This resulted in identification of a specific mRNA profile (a CD5 signature) for CD5+ DLBCL. The CD5 signature included downregulated extracellular matrix genes such as POSTN, SPARC, COL1A1, COL3A1, CTSK, MMP9 and LAMB3, and comprised upregulated genes including TRPM4. We tested this CD5 signature for its potential use as a relevant marker for CD5+ DLBCL and found that it did indeed recognize this subgroup. The tumors identified by the CD5 signature contained most of the CD5+ DLBCL cases and some CD5− DLBCL cases. Moreover, the subgroup of cases with this CD5 signature showed a poorer prognosis. The subsequent application of the CD5 signature to the analysis of an independent series of DLBCL microarray data resulted in identification of a subgroup of DLBCL cases with a similar clinical outcome, further suggesting that the CD5 signature can be used as a clinically relevant marker of this disease. (Cancer Sci 2006; 97: 868–874)

Proteinase-3 (PR3) is an abundant serine proteinase stored in the azurophilic granules of neutrophils and released to the cell surface upon activation where it contributes to local tissue destruction and inflammation. The sub-population of membrane PR3 (mPR3) high expression (PR3-high) varies among individuals. There are many reports about PR3 in Wegener's granulomatosis, but few about PR3 expression in patients with common inflammatory disorders, such as sepsis. The mPR3 expression on neutrophils from 56 patients with inflammatory disorders and from 64 healthy volunteers was examined by flow cytometry. High variability in the percentage of PR3-high (%PR3-high) neutrophils was observed in healthy volunteers and patients with inflammatory disease, and the %PR3-high was significantly greater in the patients (72 +/- 19% vs 55 +/- 20%, P0.0001). Overall neutrophil PR3 expression in patients with infectious diseases, especially systemic inflammatory response syndrome (SIRS) was significantly high (P0.01) and showed a positive correlation with C-reactive protein (CRP). Even under inflammatory conditions not involving autoimmune vasculitis, there are significant increases in both the absolute surface expression of PR3 and the numbers of neutrophils expressing high levels of PR3 and these correlate with CRP levels. The data are consistent with a model in which neutrophil membrane expression of PR3 is greatly influenced by an in vivo inflammatory environment.

Bacterial vectors may offer many advantages over other antigen delivery systems for cancer vaccines. We engineered a Salmonella typhimurium vaccine strain to deliver the NY-ESO-1 tumor antigen (S. typhimurium-NY-ESO-1) through a type III protein secretion system. The S. typhimurium-NY-ESO-1 construct elicited NY-ESO-1-specific CD8+ and CD4+ T cells from peripheral blood lymphocytes of cancer patients in vitro. Oral administration of S. typhimurium-NY-ESO-1 to mice resulted in the regression of established NY-ESO-1-expressing tumors. Intratu-moral inoculation of S. typhimurium-NY-ESO-1 to NY-ESO-1-negative tumors resulted in delivery of antigen in vivo and led to tumor regression in the presence of preexisting NY-ESO-1-spedific CD8+ T cells. Specific T cell responses against at least 2 unrelated tumor antigens not contained in the vaccine were observed, demonstrating epitope spreading. We propose that antigen delivery through the S. typhimurium type III secretion system is a promising novel strategy for cancer vaccine development.

In this study, the mRNA expression of p14 ARF in t(8;21)AML cells was found to be significantly lower than acute myelocytic leukemia (AML) cells without t(8;21) chromosome abnormality, which was concordant with previous observation by Linggi et al. that AML1-MTG8 represses the transcription of p14 ARF . Although p53 mRNA expression level of t(8;21)AML cells was not low, p53 protein expression was reduced in t(8;21)AML cells. Genotoxic damage by ionizing radiation did not induce p53 upregulation in t(8;21)AML cells. Since p14 ARF has been demonstrated to inhibit p53 degradation by binding to MDM2, repression of p14 ARF expression in t(8;21)AML may facilitate the degradation of p53 by MDM2. Low p14 ARF in t(8;21)AML may also account for the absence of upregulation of p53 by ionizing radiation. Then, we have shown that p53 expression level was inversely correlated with S/G2/M population of cell cycle in AML cells. Most of the t(8;21)AML are considered to be in p53 low S/G2/M high . It is now widely known that formation of AML1-MTG8 by t(8;21) translocation is a very early event in leukemogenesis, and AML1-MTG8 alone might have limited proliferative potential. Then, secondary oncogenic events such as activated receptor tyrosine kinase (like c-kit mutation), is necessary to become full-blown leukemia. Low p53 protein expression and insufficient induction of p53 by genotoxic damage might increase the opportunity to obtain additional oncogenic events, since genome guard function of p53 does not work in t(8;21)AML cells.

Purpose: NY-ESO-1 belongs to a class of cancer/testis antigens and has been shown to be immunogenic in cancer patients. We synthesized a complex of cholesterol-bearing hydrophobized pullulan and NY-ESO-1 protein (CHP/ESO) and investigated the in vitro stimulation of CD8 and CD4 T cells from peripheral blood mononuclear cells in healthy donors with autologous CHP/ESO-loaded dendritic cells as antigen-presenting cells.Experimental Design: In vitro stimulation of CD8 or CD4 T cells was determined by IFNγ ELISPOT assays against autologous EBV-B cells infected with vaccinia/NY-ESO-1 recombinant virus or wild-type vaccinia virus as targets and by ELISA measuring secreted IFNγ.Results: NY-ESO-1–specific CD8 and CD4 T cells were induced. In a donor expressing HLA-A2, CD8 T cells stimulated with CHP/ESO-loaded dendritic cells recognized naturally processed NY-ESO-1157-165, an HLA-A2–binding CD8 T cell epitope. NY-ESO-1 CD4 T cells were Th1-type. We identified a new HLA-DR15–binding CD4 T cell epitope, NY-ESO-137-50.Conclusions: These findings indicate that CHP/ESO is a promising polyvalent cancer vaccine targeting NY-ESO-1.

Background Since the tumor growth depends on the formation of feeding vessels, color/power Doppler ultrasonography (US) has been used to evaluate the vascular flow images of lymph nodes (LNs) in order to differentiate benign LNs from malignant LNs or lymphoma from metastatic carcinoma. Methods We performed color/power Doppler ultrasonography (US) using Levovist (LV) to evaluate the vascular patterns in the LNs of 10 patients with different types of lymphoma. The patterns were classified as central, peripheral, or avascular type. Results Vascular flow was identified in 9 of 10 LNs and it was enhanced with LV in all cases. The vascular pattern of the LNs was of the central type in all seven B-cell lymphoma patients and one of two T-cell lymphoma patients and of the peripheral type in the remaining T-cell lymphoma patient. The avascular type pattern was observed in one Hodgkin lymphoma patient even with LV. Discussion Typical vascular patterns might be associated with certain subtypes of lymphoma, and LV appears to improve the diagnostic value of Doppler US.