Search

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.

We previously found that a Salmonella typhimurium vector engineered to secrete soluble tumor antigen induces CD4+ T cells resistant to CD4+CD25+ regulatory T cells (Treg) and that glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) signal is involved in the development of this resistance. In this study, we address the potential of incorporating GITR ligand (GITRL) as a way to augment the immunogenicity of cancer vaccines. BALB/c mice were immunized by gene gun with plasmids encoding the mutated extracellular signal-regulated kinase 2 (mERK) with or without plasmids encoding mouse GITRL. Coadministration with GITRL during primary and secondary immunization enhanced the induction of mERK-specific CD8+ T cells. Antibody depletion and minigene analysis suggested that GITRL directly activated CTL epitope-specific CD8+ T cells independently of CD4+ T cells. Immunization with plasmids encoding a CTL epitope and GITRL resulted in strong tumor inhibition in a CD8+ T cell–dependent manner. Furthermore, CTL epitope-specific CD8+ T cells induced by immunization with plasmids encoding CTL epitope coadministered with GITRL were refractory to suppression by CD4+CD25+ Tregs compared with CD8+ T cells induced without GITR signaling. We propose that coadministration of GITR signaling agents with tumor antigens constitutes a promising novel strategy for cancer vaccine development. [Cancer Res 2008;68(14):5948–54]

Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, has been spreading throughout the world. To date, there are still no approved human vaccines for this disease. To develop an effective vaccine, the establishment of animal models for evaluating post-vaccination immune responses is necessary. In this study, we have identified a CTL epitope in the SARS-CoV-2 spike (S) protein that could be used to measure the cellular immune response against this protein. Potential predicted CTL epitopes of the SARS-CoV-2 S protein were investigated by immunizing BALB/c mice with a recombinant of the receptor-binding domain (RBD) of the S protein. Then, CD8+ T cells specific for S-RBD were detected by stimulating with potential epitope peptides and then measuring the interferon-gamma production. Truncation of this peptide revealed that S-RBD-specific CD8+ T cells recognized a H2-Dd-restricted S526-533 peptide. In conclusion, this animal model is suitable for evaluating the immunogenicity of SARS-CoV-2 vaccines.

We have identified a novel HLA-A*02:01-restricted CD8 T-cell epitope encompassing the H3.3K27M mutation and a corresponding high-affinity T-cell receptor (TCR) that recognizes the epitope. While the development of adoptive cell transfer therapy using TCR-transduced T-cells holds a promise, we still need to overcome multiple challenges, such as suboptimal T-cell trafficking and the immunosuppressive environment of malignant glioma. For example, tumor-associated macrophages (TAMs) mediate immunosuppression but do not function as effective antigen-presenting cells. We have developed a novel cholesteryl pullulan (CHP) nanogel as a highly biocompatible and efficient vaccine delivery system targeting TAMs. In this study, we investigated whether the CHP nanogel loaded with the H3.3K27M peptide would deliver the peptide to TAMs and convert TAMs to better antigen-presenting cells that enhance the anti- H3.3K27M+ glioma activity of the TCR-transduced T-cells. As a clinically relevant mouse model, we used HLA-A2/HLA-DR1-transgenic mice and generated a syngeneic glioma cell line that expresses H3.3K27M from their astrocytes. We also generated a retroviral vector encoding the H3.3K27M-specific TCR for transduction of mouse T cells. HLA-A2/HLA-DR1-transgenic mice bearing day 16 intracerebral H3.3K27M+ glioma received an intravenous administration of the CHP nanogel along with poly-ICLC, a Toll-like receptor 3 agonist. The mice then received an intravenous infusion of TCR-transduced or control, non-transduced T-cells on the following day. The triple combination regimen with the CHP, poly-ICLC and TCR-transduced T-cells significantly suppressed the tumor growth, associated with increased levels of T-cell infiltration into the tumors compared with the dual-therapy with poly-ICLC and TCR-T-cells without the CHP. Furthermore, TAMs isolated from CHP-treated mice showed evidence of CHP-uptake, abilities to stimulate proliferation of TCR-transduced T-cells, and higher levels of HLA.A2 expression. These results suggest that the antigen-loaded CHP nanogel can promote the local antigen-presentation to T-cells and represent a promising approach for improving the efficacy of adoptive T-cell therapy for gliomas.

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.

Immune checkpoint inhibitors and adoptive transfer of gene-engineered T cells have emerged as novel therapeutic modalities for hard-to-treat solid tumors; however, many patients are refractory to these immunotherapies, and the mechanisms underlying tumor immune resistance have not been fully elucidated. By comparing the tumor microenvironment of checkpoint inhibition-sensitive and -resistant murine solid tumors, we observed that the resistant tumors had low immunogenicity. We identified antigen presentation by CD11b+F4/80+ tumor-associated macrophages (TAMs) as a key factor correlated with immune resistance. In the resistant tumors, TAMs remained inactive and did not exert antigen-presenting activity. Targeted delivery of a long peptide antigen to TAMs by using a nano-sized hydrogel (nanogel) in the presence of a TLR agonist activated TAMs, induced their antigen-presenting activity, and thereby transformed the resistant tumors into tumors sensitive to adaptive immune responses such as adoptive transfer of tumor-specific T cell receptor-engineered T cells. These results indicate that the status and function of TAMs have a significant impact on tumor immune sensitivity and that manipulation of TAM functions would be an effective approach for improving the efficacy of immunotherapies., The Journal of clinical investigation, 129(3), pp.1278-1294; 2019

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.

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 was oesophageal cancer in this study. 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 positivity. Our data indicate that oesophageal cancer is a promising candidate tumour for CT antigen-targeting immunotherapy and immune monitoring.

Additional file 2 Figure 2. CT antigen mRNA expression in normal tissue. mRNA expression of MAGE-A4 (A), NY-ESO-1 (B) and SAGE (C) in normal tissue was shown. First Choice™ Human Total RNA Survey Panel®, Human Breast Total RNA®, Human Lymph node Total RNA®, Human Testicle Total RNA® and Human Uterus Total RNA® (Ambion KK, Tokyo, Japan) were used.

Additional file 3 Table 1. Median levels of CT antigen mRNAs in each CT antigen-positive tumour type.

Additional file 1 Figure 1. Relationship among MAGE-A4, NY-ESO-1 and SAGE mRNA expression in oesophageal cancer and other cancer types. Pearson’s chi-squared test of independence was used for evaluation. In oesophageal cancer, there was a relationship among MAGE-A4, NY-ESO-1 and SAGE expression (A-C, left; all p

Additional file 4 Table 2. MAGE-A4 and NY-ESO-1 IHC analyses of mRNA-assessed tumour samples.

// 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.

MAGE-A4 antigen is a cancer-testis antigen that is frequently expressed in tumor tissues. Cholesteryl pullulan (CHP) is a novel antigen delivery system for cancer vaccines. This study evaluated the safety, immune responses and clinical outcomes of patients who received a CHP-MAGE-A4 vaccine. Twenty-two patients with advanced or metastatic cancer were enrolled, and were subcutaneously vaccinated with either 100 μg or 300 μg of CHP-MAGE-A4. Seven and 15 patients, respectively, were repeatedly vaccinated with 100 μg or 300 μg of CHP-MAGE-A4; patients in both groups received a median of 7 doses. No serious adverse events related to the vaccine were observed. Of 7 patients receiving the 100 μg dose, 2 (29%) showed immune responses, compared with 3 of the 14 (21%) patients who received the 300 μg dose. In total, MAGE-A4-specific antibody responses were induced in 5 of 21 (24%) patients. No differences in survival were seen between patients receiving the 100 μg and 300 μg doses, or between immune responders and non-responders. Eleven (50%) patients had pre-existing antibodies to NY-ESO-1. In 16 patients with esophageal or head/neck squamous cell carcinoma, the survival time was significantly shorter in those who had NY-ESO-1-co-expressing tumors. Patients with high pre-existing antibody responses to NY-ESO-1 displayed worse prognosis than those with no pre-existing response. Therefore, in planning clinical trials of MAGE-A4 vaccine, enrolling NY-ESO-1-expressing tumor or not would be a critical issue to be discussed. Combination vaccines of MAGE-A4 and NY-ESO-1 antigens would be one of the strategies to overcome the poor prognosis.

Objectives To evaluate pregnancy outcomes in Japanese patients with multiple sclerosis (MS) enrolled in a postmarketing surveillance study of intramuscular interferon beta-1a (IM IFN beta-1a). Methods Safety data were collected from Japanese patients receiving 30 μg weekly IM IFN beta-1a. Pregnancy outcomes and annualized relapse rates (ARR) were analyzed retrospectively in patients who registered into the postmarketing surveillance study. Results A total of 1110 of 1638 patients registered in the postmarketing surveillance study were women. A total of 21 pregnancies (20 patients) resulted in 17 live births, 2 induced abortions, one spontaneous abortion and one unknown outcome. Weights and lengths of the 17 newborns were similar to newborns in the general Japanese population. No complications or malformations were reported. Of these 20 patients, nine experienced relapses in the year after childbirth, two experienced relapses in the year before pregnancy, and one experienced relapses before and during pregnancy. The mean (standard deviation) ARR was 0.94 (2.18) in the year before pregnancy, 0.25 (1.00), 0 (0) and 0 (0) in the three trimesters of pregnancy, and 1.05 (1.81), 0.84 (1.68), 0.63 (2.01) and 0.21 (0.92) in the first four quarters postpartum. Of the five patients who relapsed during the first quarter postpartum, only one had resumed IFN beta-1a treatment for 35 days. Conclusions Although sample size limits our ability to draw definitive conclusions, we did not find evidence that IFN beta-1a has adverse effects on pregnancy outcomes in Japanese patients with MS. Early IFN beta-1a resumption might reduce the risk of relapse within the first quarter postpartum.

Because existing therapeutic cancer vaccines provide only a limited clinical benefit, a different vaccination strategy is necessary to improve vaccine efficacy. We developed a nanoparticulate cancer vaccine by encapsulating a synthetic long peptide antigen within an immunologically inert nanoparticulate hydrogel (nanogel) of cholesteryl pullulan (CHP). After subcutaneous injection to mice, the nanogel-based vaccine was efficiently transported to the draining lymph node, and was preferentially engulfed by medullary macrophages but was not sensed by other macrophages and dendritic cells (so-called "immunologically stealth mode"). Although the function of medullary macrophages in T cell immunity has been unexplored so far, these macrophages effectively cross-primed the vaccine-specific CD8(+) T cells in the presence of a Toll-like receptor (TLR) agonist as an adjuvant. The nanogel-based vaccine significantly inhibited in vivo tumor growth in the prophylactic and therapeutic settings, compared to another vaccine formulation using a conventional delivery system, incomplete Freund's adjuvant. We also revealed that lymph node macrophages were highly responsive to TLR stimulation, which may underlie the potency of the macrophage-oriented, nanogel-based vaccine. These results indicate that targeting medullary macrophages using the immunologically stealth nanoparticulate delivery system is an effective vaccine strategy.

TPS11074 Background: Combination therapy to enhance the efficacy of T cell receptor (TCR)-engineered T cells (TCR-T) has received increasing attention. We found that a combination therapy of TCR-T and a long peptide vaccine with CpG adjuvant without lymphodepletion regimen caused regression of immune checkpoint inhibitor-resistant sarcoma in a preclinical mouse model. Based on this finding, we initiated a clinical trial of TBI-1301 combined with CHP:NE1 without lymphodepletion for the patients with NY-ESO-1-expressing advanced soft tissue sarcoma. TBI-1301 is an NY-ESO-1157–165/HLA-A*02:01- or -A*02:06-specific TCR-T engineered to reduce endogenous TCR mRNA expression. CHP:NE1 is a novel T cell stimulator consisting of NY-ESO-1 long peptide antigen, cholesteryl pullulan (CHP) nanogel, and CpG oligoDNA. CHP nanogel is used for efficient delivery of long peptide antigen into the lymph nodes. CpG oligoDNA is a TLR9 agonist and used as an adjuvant. CHP:NE1 is expected to reinforce TBI-1301 T cells in the lymph nodes. Methods: This is an investigator-initiated multi-institutional first-in-human phase I/II clinical trial. TBI-1301 is infused at 5×109 cells one day after subcutaneous injection of CHP:NE1. CHP:NE1 is injected again 7 days after TBI-1301 infusion. This cycle is repeated once more. Lymphodepletion using cyclophosphamide and/or fludarabine is not performed. Key inclusion criteria include: refractory soft tissue sarcoma with NY-ESO-1 antigen expression, HLA-A*02:01- or HLA-A*02:06- positive, ECOG Performance Status 0 or 1, and adequate organ function. The primary objective is to assess the safety and efficacy in the phase 1 and 2 parts, respectively. The secondary objective is to assess the efficacy and immune response (blood concentration, immunophenotype and activity of TBI-1301) in the phase 1 part and the safety and immune response in the phase 2 part. Enrollment in this study is currently ongoing. Clinical trial information: JMA-IIA00346.

The CXCR3 ligands CXCL9, 10, and 11 play critical roles in the amplification of immune responses by recruiting CXCR3+ immune effector cells to the tumor site. Taking advantage of this property of CXCR3 ligands, we aimed to establish a novel approach to identify immunogenic mutated-antigens. We examined the feasibility of using CXCR3 ligand mRNAs as sensors for detection of specific immune responses in human and murine systems. We further investigated whether this approach is applicable for the identification of immunogenic mutated-antigens by using murine sarcoma lines. Rapid synthesis of CXCR3 ligand mRNAs occurred shortly after specific immune responses in both human and murine immune systems. Particularly, in CMS5 tumor-bearing mice, we detected specific immune responses to mutated mitogen-activated protein kinase 2 (ERK2), which has previously been identified as an immunogenic mutated-antigen. Furthermore, by combining this approach with whole-exome and transcriptome sequencing analyses, we identified an immunogenic neo-epitope derived from mutated staphylococcal nuclease domain-containing protein 1 (Snd1) in CMS7 tumor-bearing mice. Most importantly, we successfully detected the specific immune response to this neo-epitope even without co-administration of anti-cytotoxic T-lymphocyte protein-4 (CTLA-4), anti-programmed cell death-1 (PD-1) and anti-glucocorticoid-induced TNFR-related protein (GITR) antibodies, which vigorously augmented the immune response and consequently enabled us to detect the specific immune response to this neo-epitope by conventional IFNγ intracellular staining method. Our data indicate the potential usefulness of this strategy for the identification of immunogenic mutated-antigens. We propose that this approach would be of great help for the development of personalized cancer vaccine therapies in future.

CD8+ CTLs play a critical role in antitumor immunity. However, vaccination with synthetic peptide containing CTL epitopes has not been generally effective in inducing protective antitumor immunity. In this study, we addressed the detailed mechanism(s) involved in this failure using a new tumor model of BALB/c transplanted tumors expressing NY-ESO-1, an extensively studied human cancer/testis Ag. Whereas peptide immunization with an H2-Dd–restricted CTL epitope derived from NY-ESO-1 (NY-ESO-1 p81–88) induced NY-ESO-181–88–specific CD8+ T cells in draining lymph nodes and spleens, tumor growth was significantly enhanced. Single-cell analysis of specific CD8+ T cells revealed that peptide immunization caused apoptosis of >80% of NY-ESO-181–88–specific CD8+ T cells at tumor sites and repetitive immunization further diminished the number of specific CD8+ T cells. This phenomenon was associated with elevated surface expression of Fas and programmed death-1. When peptide vaccination was combined with an adjuvant, a TLR9 ligand CpG, the elevated Fas and programmed death-1 expression and apoptosis induction were not observed, and vaccine with peptide and CpG was associated with strong tumor growth inhibition. Selection of appropriate adjuvants is essential for development of effective cancer vaccines, with protection of effector T cells from peptide vaccine-induced apoptosis being a prime objective.

e15176Background: Complexes of Cholesteryl pullulan and NY-ESO-1 antigen (CHP-NY-ESO-1) present multiple epitope peptides to both the MHC class I and class II pathways. MIS416 is a non-toxic microp...

We previously found that a Salmonella typhimurium vector engineered to secrete soluble tumor antigen induces CD4+ T cells resistant to CD4+CD25+ regulatory T cells (Treg) and that glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) signal is involved in the development of this resistance. In this study, we address the potential of incorporating GITR ligand (GITRL) as a way to augment the immunogenicity of cancer vaccines. BALB/c mice were immunized by gene gun with plasmids encoding the mutated extracellular signal-regulated kinase 2 (mERK) with or without plasmids encoding mouse GITRL. Coadministration with GITRL during primary and secondary immunization enhanced the induction of mERK-specific CD8+ T cells. Antibody depletion and minigene analysis suggested that GITRL directly activated CTL epitope-specific CD8+ T cells independently of CD4+ T cells. Immunization with plasmids encoding a CTL epitope and GITRL resulted in strong tumor inhibition in a CD8+ T cell–dependent manner. Furthermore, CTL epitope-specific CD8+ T cells induced by immunization with plasmids encoding CTL epitope coadministered with GITRL were refractory to suppression by CD4+CD25+ Tregs compared with CD8+ T cells induced without GITR signaling. We propose that coadministration of GITR signaling agents with tumor antigens constitutes a promising novel strategy for cancer vaccine development. [Cancer Res 2008;68(14):5948–54]

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.

Objectives To evaluate the efficacy and safety of 2 years of treatment with intramuscular interferon beta-1a (IM IFN beta-1a) in Japanese patients with relapsing-remitting multiple sclerosis, with an exploratory analysis of the impact of initial dose titration on tolerability. Methods Japanese patients with relapsing-remitting multiple sclerosis were randomized to receive IM IFN beta-1a at dosages of either 30mcg once weekly (full-dose group, n =50) or 15mcg once weekly for 2 weeks then 30mcg once weekly thereafter (titration group, n =50). Key outcomes included annualized relapse rate (ARR) at 2 years (primary endpoint), change in disability measured using the Expanded Disability Status Scale (EDSS), safety, and tolerability. Results The ARR (95% CI) decreased from 1.540 (1.381–1.718) at baseline to 0.371 (0.240–0.571) at Year 1 and 0.351 (0.244–0.503) at Year 2. EDSS improvements were apparent from Week 24; the mean change from baseline EDSS score (2.1) at Year 2 was −0.34 ( P =0.004). The most frequently reported adverse events were influenza-like illness (92%), nasopharyngitis (57%), relapse of multiple sclerosis (51%), and injection-site reaction (30%). The overall incidence and severity of influenza-like symptoms were similar in the full-dose group and titration group; only 1 participant, in the full-dose group (2%), experienced severe influenza-like symptoms. However, the incidence of influenza-like symptoms was slightly reduced at earlier timepoints in the titration group. Conclusions The results of this 2-year study demonstrate that IM IFN beta-1a can be used effectively and safely in Japanese patients with relapsing-remitting multiple sclerosis for an extended period of time.

To utilize heat generated from high-temperature processes, the kinetic feasibility of a calcium oxide/lead oxide/carbon dioxide chemical heat pump was examined experimentally by kinetic studies of CaO/CO2 and PbO/CO2 reaction systems, which constitute the heat pump’s reaction. In order to determine the optimal reaction conditions that still allow practical operation of the heat pump, both reaction systems were examined with respect to thermal drivability and reaction material durability. The heat pump was able to store heat of about 860°C and transform it to a heat of above 880°C under sub-atmospheric pressure without mechanical work. An applied system that combined the heat pump with a high-temperature process was proposed for high-efficiency heat utilization. The scale of the heat pump in the combined system was estimated from the experimental results.

A new chemical heat pump designed to utilize high-temperature heat above 800°C generated from a high-temperature gas nuclear reactor or other high-temperature industrial process is presented. Based on experimental results, a heat pump that uses a calcium oxide / lead oxide I carbon dioxide reaction system appears to be a suitable system. To demonstrate the validity of the heat pump, the equilibrium relationship and kinetics of calcium oxide I carbon dioxide and lead oxide I carbon dioxide, which comprise the reaction system of the heat pump, are studied experimentally. A study of the equilibrium relationship of lead oxide / carbon dioxide, which consists of a three-step equilibrium, reveals that the highest temperature equilibrium relationship of the three-step equilibrium is associated with optimal heat pump operation. The practical operation conditions of the heat pump are determined based on the equilibrium relationship and kinetic experiments. The proposed heat pump may be able to operate as a heat transformation-type heat pump, and is capable of storing heat above approximately 830°C and transforming the heat to a higher temperature of more than 870°C under subatmospheric pressure and thermal driving conditions with no mechanical work. The calculated mean heat output and heat output amount are 670 W/kg-CaCO 3 and 1200 kJ/kg-CaCO 3 , respectively, at 870°C, 1 atm for 30 min. Thus, the new heat pump can be applied to a heat storage and heat transformation system for high temperature processes.

The dynamic rearrangement of cell–cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell–cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell–cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell–cell contacts and found that AF-6 accumulated at the cell–cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell–cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell–cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell–cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell–cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.

Ras (Ha-Ras, Ki-Ras, N-Ras) is implicated in the regulation of various cell functions such as gene expression and cell proliferation downstream from specific extracellular signals. Here, we partially purified a Ras-interacting protein with molecular mass of about 180 kDa (p180) from bovine brain membrane extract by glutathione S-transferase (GST)-Ha-Ras affinity column chromatography. This protein bound to the GTP gamma S (guanosine 5'-(3-O-thio)triphosphate, a nonhydrolyzable GTP analog).GST-Ha-Ras affinity column but not to those containing GDP.GST-Ha-Ras or GTP gamma S.GST-Ha-Ras with a mutation in the effector domain (Ha-RasA38). The amino acid sequences of the peptides derived from p180 were almost identical to those of human AF-6 that is identified as the fusion partner of the ALL-1 protein. The ALL-1/AF-6 chimeric protein is the critical product of the t (6:11) abnormality associated with some human leukemia. AF-6 has a GLGF/Dlg homology repeat (DHR) motif and shows a high degree of sequence similarity with Drosophila Canoe, which is assumed to function downstream from Notch in a common developmental pathway. The recombinant N-terminal domain of AF-6 and Canoe specifically interacted with GTP gamma S.GST-Ha-Ras. The known Ras target c-Raf-1 inhibited the interaction of AF-6 with GTP gamma S.GST-Ha-Ras. These results indicate that AF-6 and Canoe are putative targets for Ras.

Recently, immune checkpoint inhibitors using anti-CTLA-4, anti-PD-1, and anti-PD-L1 antibodies has been emerging as novel, effective modality for the treatment of cancer. However, immune checkpoint inhibitors have been proven to be effective only for a partial group of patients who have lymphocyte infiltration or inflammation in the tumor microenvironment prior to the treatment. Although the relationship between efficacy of checkpoint inhibitors and anti-tumor cellular immune response has been revealed, these studies paid attention only for the induction of immune responses in lymph node and do not pay attention to the mechanisms regulating the anti-tumor cellular immune responses in tumor site. In this study, by using checkpoint inhibitors-sensitive or resistant murine in vivo tumors, we explored a mechanism underlying the resistance focusing on immune cells at the tumor site. We newly identified CD11b+/F4/80+ tumor-associated macrophages as a key factor highly correlated to the resistance. In the resistant tumor, tumor-associated macrophages remained immature and did not exert antigen-presenting activity. A nanogel-based antigen delivery system enabled to selectively and efficiently deliver a long peptide antigen to tumor-associated macrophages. Co-administration of the nanogel:long peptide antigen and TLR agonist activated tumor-associated macrophages and induced antigen presentation by these cells. This change in the tumor microenvironment turned the resistant tumor to treatment-sensitive, in particular, adoptive transfer of TCR-engineered T cells after the treatment with nanogel:long peptide antigen and TLR agonist resulted in the cure of resistant tumor. These results indicate that the status and function of tumor-associated macrophages have a great impact on treatment sensitivity, and that these cells might be a promising target to improve the efficacy of immunotherapy including checkpoint inhibitors and adoptive cell therapies. This abstract is also being presented as Poster B68. Citation Format: Daisuke Muraoka, Naozumi Harada, Naohiro Seo, Tae Hayashi, Keisuke Fujii, Rui Yamaguchi, Seiya Imoto, Satoru Miyano, Hideo Yagita, Kazunari Akiyoshi, Hiroshi Shiku. Antigen delivery targeting tumor-infiltrating macrophages leads to eradication of tumor highly resistant to immune checkpoint inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR02.

Many patients develop tumor antigen-specific T cell responses detectable in peripheral blood mononuclear cells (PBMCs) following cancer vaccine. However, measurable tumor regression is observed in a limited number of patients receiving cancer vaccines. There is a need to re-evaluate systemically the immune responses induced by cancer vaccines. Here, we established animal models targeting two human cancer/testis antigens, NY-ESO-1 and MAGE-A4. Cytotoxic T lymphocyte (CTL) epitopes of these antigens were investigated by immunizing BALB/c mice with plasmids encoding the entire sequences of NY-ESO-1 or MAGE-A4. CD8 + T cells specific for NY-ESO-1 or MAGE-A4 were able to be detected by ELISPOT assays using antigen presenting cells pulsed with overlapping peptides covering the whole protein, indicating the high immunogenicity of these antigens in mice. Truncation of these peptides revealed that NY-ESO-1-specific CD8 + T cells recognized D d -restricted 8mer peptides, NY-ESO-1 81-88 . MAGE-A4-specific CD8 + T cells recognized D d -restricted 9mer peptides, MAGE-A4 265-273 . MHC/peptide tetramers allowed us to analyze the kinetics and distribution of the antigen-specific immune responses, and we found that stronger antigen-specific CD8 + T cell responses were required for more effective anti-tumor activity. Taken together, these animal models are valuable for evaluation of immune responses and optimization of the efficacy of cancer vaccines.

がんと免疫に関する研究の進展を背景に，現在がん治療のためのがんワクチンの開発が世界的に進められている．我々は，がんに対する獲得免疫におけるキラーT細胞，ヘルパーT細胞および抗原提示細胞の重要性に注目して設計した新しい多価性がんワクチン「CHPがんタンパク質ワクチン」の開発を進めている．本ワクチンはキラーT細胞とヘルパーT細胞の同時活性化を達成するために全長の抗原タンパク質を用い，抗原タンパク質の抗原提示細胞への効率的な送達とクロスプレゼンテーションを促す新規抗原デリバリーシステムCHPを取り入れている点を特徴としている．本ワクチンは臨床・非臨床・GMP製造の各ステップにおけるアカデミアと企業の協奏的な努力の末に現在，実用化を目指して国内外で治験が進められている．

Telomerase is the enzyme that elongates telomere repeat at the ends of a chromosome. As high telomerase activity is observed in most cancer cells, inhibitors of human telomerase have been expected as new chemotherapeutic agents for cancer. We describe here the discovery of novel inhibitors with IC50 values in the submicromolar range. The structure of the novel inhibitors will be useful as a scaffold for construction of the library in the search for telomerase inhibitors.

CHP-NY-ESO-1 is a novel therapeutic cancer vaccine consisting of a recombinant protein of cancer antigen NY-ESO-1 and a polysaccharide-based delivery system, cholesteryl pullulan. A pilot clinical study of CHP-NY-ESO-1 in cancer patients was previously conducted, and the adverse events related to this drug were observed to be limited to skin reactions at injection sites. To further establish the safety of CHP-NY-ESO-1, we studied the effects of its subcutaneous injection on vital functions such as the central nervous system, cardiovascular system and respiratory system using preclinical animal models. The effects of CHP-NY-ESO-1 on the cardiovascular system were investigated in dogs using a telemetry system for blood pressure and heart rate and the Holter monitoring for ambulatory electrocardiograms. No drug-related changes were observed in these parameters. The effect of CHP-NY-ESO-1 on the hERG-dependent potassium currents was also examined using in vitro cultured cell system, and no inhibition of hERG currents was observed. The effects of CHP-NY-ESO-1 on the central nervous system were examined in rats using functional observational battery method, and no drug-related changes were observed in home cage observations, open field observations, hand held observations, and perception and motor function observations. The effect of CHP-NY-ESO-1 on the respiratory system was investigated in rats by measuring tidal volume, minute volume and respiratory rate using whole-body plethysmograph method, and no significant changes were found in these parameters. These results indicate that CHP-NY-ESO-1 would not have any pharmacological effects on vital functions and support the safety of this cancer vaccine for clinical use.

Purpose: Memory CD8+ T cells characterized the long survival after the strong activation followed by the appropriate antigenic stimulation have been believed to be an effective tumoricidal effectors in patients with cancer because they are possible to attack tumor cells repeatedly for a long time. Despite its importance, analysis of differentiation and function of memory CD8+ T cells is insufficient. In this study, we compared the memory CD8+ T cells induced by two murine vaccine systems to identify mechanism(s) participating in maintenance of effector function of tumoricidal long survival memory CTLs. Methods and Results: Full-length human NY-ESO-1 cDNA or CTL epitope peptide of NY-ESO-1 (p81-88) was immunized subcutaneously (s.c.) in BALB/c mice twice at 2 weeks interval or 3 times at weekly interval, respectively. At 44 days after the final vaccination, NY-ESO-1-expressing CT26 cells (CT26/NY-ESO-1) were inoculated s.c. of immunized mice, and subsequent tumor growth was measured. In addition, IFN-γ-expressing CD8+ T cell ratio in spleen of the immunized mice was monitored to evaluate vaccination efficacy. As a result, the inhibition of tumor growth was surprisingly shown in the NY-ESO-1 cDNA vaccination group more vigorously than the NY-ESO-1 CTL epitope peptide-treated group in comparison with untreated group. In addition, significant elevation of vaccine-specific IFN-γ-expressing CD8+ T cell ratio was observed in spleen of the NY-ESO-1 cDNA-treated mice. These results indicated that quality of memory CTLs was fully maintained in DNA vaccination group, but not peptide-treated group. To address this potent issue of superiority of DNA vaccination on the generation of long survival memory CTLs, vaccine-specific CTLs from DNA and peptide vaccination groups were purified and subjected to the total RNA micro array analysis. Memory CTLs from DNA-treated mice expressed signal transducing adaptor protein 2 (STAP2) over a 200 fold change compared with peptide-induced CTLs. Moreover, we clearly demonstrated by the study using STAP2 siRNA that STAP2 is essential for the maintenance of the tumoricidal effector function and cytokine expression of DNA-induced long survival memory CTLs. Conclusion: In this study, our murine vaccination system using human NY-ESO-1 gene and peptide clearly indicated that DNA vaccination is effective for the generation of tumoricidal long survival memory CTLs. In addition, we demonstrated for the first time that STAP2 plays as a crucial factor in the long-term maintenance of memory CTLs by probably the lasting care of cytokine expression and cytotoxicity. Since the maintenance of the effector function of the tumor antigen-specific memory CTLs has been thought to influence directly in the outcome of caner immunotherapy, our results regarding correlation between the generation of long survival memory CTLs and STAP2 have a possibility to be useful in the future treatment of patients with cancer. Citation Format: Daisuke Muraoka, Naohiro Seo, Tae Hayashi, Naozumi Harada, Hiroshi Shiku. Signal transducing adaptor protein 2 (STAP2) has the crucial role in maintaining the CTL function of memory T cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B34.

Telomerase inhibitors are expected as a new candidate of therapeutic agents for cancer. Recently, we have found novel inhibitors based on the bisindole skeleton. In this study, solid-phase synthesis was applied to construct a library of inhibitors having aromatic phosphate, long alkyl chain and tryptophane components, from which a d , d -ditryptophane derivative has been identified as a new potent telomerase inhibitor with IC 50 values of 0.3 μM. A hypothetical binding model for the new inhibitors has been proposed based on the structure–activity relationship.

Telomerase is the enzyme that elongates telomere repeat at the ends of a chromosome. As high telomerase activity is observed in most cancer cells, inhibitors of human telomerase have been expected as new chemotherapeutic agents for cancer. We describe here the discovery of novel inhibitors with IC50 values in the submicromolar range. The structure of the novel inhibitors will be useful as a scaffold for construction of the library in the search for telomerase inhibitors.

AF-6 contains two putative Ras-associating domains (RA domains) which are seen in several Ras effectors such as RalGDS and RIN1. We previously showed that an AF-6 fragment containing the amino-terminal (N-terminal) RA domain directly binds to activated Ras and ZO-1 in vitro. In this study, we showed that a single amino acid mutation in the N-terminal RA domain of AF-6 abolished the interaction of AF-6 with activated Ras and that the sites of this critical amino acid residue were similar to those for Raf-1 and RalGDS. The overexpression of the N-terminal RA domain of AF-6 inhibited the Ras-dependent c- fos promoter/enhancer stimulation in NIH3T3 cells. Endogenous AF-6 was coimmunoprecipitated with activated Ras from Rat1 cells expressing activated Ras. Moreover, we showed that AF-6 was coimmunoprecipitated with ZO-1 from Rat1 cells. Taken together, these results indicate that the Ras-interacting region on AF-6 is structurally similar to that on Raf-1 and on RalGDS and that AF-6 interacts with activated Ras and ZO-1 in vivo.