A phase 1 trial of NY-ESO-1-specific TCR-engineered T-cell therapy combined with a lymph node-targeting nanoparticulate peptide vaccine for the treatment of advanced soft tissue sarcoma.

The efficacy of immune checkpoint inhibitors is limited in refractory solid tumors. T-cell receptor gene-modified T (TCR-T)-cell therapy has attracted attention as a new immunotherapy for refractory cold tumors. We first investigated the preclinical efficacy and mode of action of TCR-T cells combined with the pullulan nanogel:long peptide antigen (LPA) vaccine in a mouse sarcoma model that is resistant to immune checkpoint inhibition. Without lymphodepletion, the pullulan nanogel:LPA vaccine markedly increased the number of TCR-T cells in the draining lymph node and tumor tissue. This change was associated with enhanced CXCR3 expression in TCR-T cells in the draining lymph node. In the phase 1 trial, autologous New York esophageal squamous cell carcinoma 1 (NY-ESO-1)-specific TCR-T cells were infused twice into HLA-matched patients with NY-ESO-1+ soft tissue sarcoma (STS). The pullulan nanogel:LPA vaccine contains an epitope recognized by TCR-T cells, and it was subcutaneously injected 1 day before and 7 days after the infusion of TCR-T cells. Lymphodepletion was not performed. Three patients with refractory synovial sarcoma (SS) were treated. Two out of the three patients developed cytokine release syndrome (CRS) with low-to-moderate cytokine level elevation. We found obvious tumor shrinkage lasting for more than 2 years by tumor imaging and long-term persistence of TCR-T cells in one patient. In conclusion, NY-ESO-1-specific TCR-T-cell therapy plus vaccination with the pullulan nanogel carrying an LPA containing the NY-ESO-1 epitope without lymphodepletion is feasible and can induce promising long-lasting therapeutic effects in refractory SS (Registration ID: JMA-IIA00346).

First-in-human phase I clinical trial of the NY-ESO-1 protein cancer vaccine with NOD2 and TLR9 stimulants in patients with NY-ESO-1-expressing refractory solid tumors.

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.

MAGE-A4, NY-ESO-1 and SAGE mRNA expression rates and co-expression relationships in solid tumours.

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.

Dose-dependent effects of NY-ESO-1 protein vaccine complexed with cholesteryl pullulan (CHP-NY-ESO-1) on immune responses and survival benefits of esophageal cancer patients.

BACKGROUND: 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 the CHP-NY-ESO-1 complex vaccine, Drug code: IMF-001. METHODS: Patients with advanced/metastatic esophageal cancer were enrolled and subcutaneously vaccinated with either 100 µg or 200 µg of NY-ESO-1 protein complexed with CHP. The primary endpoints were safety and humoral immune responses, and the secondary endpoint was clinical efficacy. RESULTS: A total of 25 patients were enrolled. Thirteen and twelve patients were repeatedly vaccinated with 100 µg or 200 µg of CHP-NY-ESO-1 with a median of 8 or 9.5 doses, respectively. No serious adverse events related to the vaccine were observed. Three out of 13 patients in the 100-µg cohort and 7 out of 12 patients in the 200-µg cohort were positive for anti-NY-ESO-1 antibodies at baseline. In the 100-µg cohort, an antibody response was observed in 5 out of 10 pre-antibody-negatives patients, and the antibody levels were augmented in 2 pre-antibody-positive patients after vaccination. In the 200-µg cohort, all 5 pre-antibody-negative patients became seropositive, and the antibody level was amplified in all 7 pre-antibody-positive patients. No tumor shrinkage was observed. The patients who received 200 µg of CHP-NY-ESO-1 survived longer than patients receiving 100 µg of CHP-NY-ESO-1, even those who exhibited unresponsiveness to previous therapies or had higher tumor burdens. CONCLUSIONS: The safety and immunogenicity of CHP-NY-ESO-1 vaccine were confirmed. The 200 µg dose more efficiently induced immune responses and suggested better survival benefits. (Clinical trial registration number NCT01003808).

Peptide vaccine induces enhanced tumor growth associated with apoptosis induction in CD8+ T cells.

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-D(d)-restricted CTL epitope derived from NY-ESO-1 (NY-ESO-1 p81-88) induced NY-ESO-1(81-88)-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-1(81-88)-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.

Self-assembled polysaccharide nanogel delivery system for overcoming tumor immune resistance.

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.

Identification of a dominant CD8+ CTL epitope in the SARS-associated coronavirus 2 spike protein.

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.

Antigen delivery targeted to tumor-associated macrophages overcomes tumor immune resistance.

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.

NY-ESO-1 antigen expression and immune response are associated with poor prognosis in MAGE-A4-vaccinated patients with esophageal or head/neck squamous cell carcinoma.

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.

Activated CD8+ T cell extracellular vesicles prevent tumour progression by targeting of lesional mesenchymal cells.

Fibroblastic tumour stroma comprising mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) promotes the invasive and metastatic properties of tumour cells. Here we show that activated CD8+ T cell-derived extracellular vesicles (EVs) interrupt fibroblastic stroma-mediated tumour progression. Activated CD8+ T cells from healthy mice transiently release cytotoxic EVs causing marked attenuation of tumour invasion and metastasis by apoptotic depletion of mesenchymal tumour stromal cells. Infiltration of EV-producing CD8+ T cells is observed in neovascular areas with high mesenchymal cell density, and tumour MSC depletion is associated with preferential engulfment of CD8+ T cell EVs in this setting. Thus, CD8+ T cells have the capacity to protect tumour progression by EV-mediated depletion of mesenchymal tumour stromal cells in addition to their conventional direct cytotoxicity against tumour cells.

Signal-transducing adaptor protein-2 promotes generation of functional long-term memory CD8+ T cells by preventing terminal effector differentiation.

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.

Identification of an immunogenic neo-epitope encoded by mouse sarcoma using CXCR3 ligand mRNAs as sensors.

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.

Long-term efficacy and safety of intramuscular interferon beta-1a: Randomized postmarketing trial of two dosing regimens in Japanese patients with relapsing-remitting multiple sclerosis.

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.

Guanine-Rich Sequences Are a Dominant Feature of Exosomal microRNAs across the Mammalian Species and Cell Types.

Exosome is an extracellular vesicle released from multivesicular endosomes and contains micro (mi) RNAs and functional proteins derived from the donor cells. Exosomal miRNAs act as an effector during communication with appropriate recipient cells, this can aid in the utilization of the exosomes in a drug delivery system for various disorders including malignancies. Differences in the miRNA distribution pattern between exosomes and donor cells indicate the active translocation of miRNAs into the exosome cargos in a miRNA sequence-dependent manner, although the molecular mechanism is little known. In this study, we statistically analyzed the miRNA microarray data and revealed that the guanine (G)-rich sequence is a dominant feature of exosome-dominant miRNAs, across the mammalian species-specificity and the cell types. Our results provide important information regarding the potential use of exosome cargos to develop miRNA-based drugs for the treatment of human diseases.

Epirubicin, Identified Using a Novel Luciferase Reporter Assay for Foxp3 Inhibitors, Inhibits Regulatory T Cell Activity.

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.

Engineering hybrid exosomes by membrane fusion with liposomes.

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.

Nanogel-based immunologically stealth vaccine targets macrophages in the medulla of lymph node and induces potent antitumor immunity.

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.

Establishment of animal models to analyze the kinetics and distribution of human tumor antigen-specific CD8⁺ T cells.

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-181-88. MAGE-A4-specific CD8(+) T cells recognized D(d)-restricted 9mer peptides, MAGE-A4265-273. 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.

Preclinical safety pharmacology study of a novel protein-based cancer vaccine CHP-NY-ESO-1.

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.

Regulatory T cell-resistant CD8+ T cells induced by glucocorticoid-induced tumor necrosis factor receptor signaling.

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.