Terukazu Kuramoto

Immunologic Evaluation of Personalized Peptide Vaccination for Patients with Advanced Malignant Glioma

Purpose: The primary goal of this phase I study was to assess the safety and immunologic responses of personalized peptide vaccination for patients with advanced malignant glioma. Experimental Design: Twenty-five patients with advanced malignant glioma (8 grade 3 and 17 grade 4 gliomas) were evaluated in a phase I clinical study of a personalized peptide vaccination. For personalized peptide vaccination, prevaccination peripheral blood mononuclear cells and plasma were provided to examine cellular and humoral responses to 25 or 23 peptides in HLA-A24+ or HLA-A2+ patients, respectively; then, only the reactive peptides (maximum of four) were used for in vivo administration. Results: The protocols were well tolerated with local redness and swelling at the injection site in most cases. Twenty-one patients received more than six vaccinations and were evaluated for both immunologic and clinical responses. Increases in cellular or humoral responses specific to at least one of the vaccinated peptides were observed in the postvaccination (sixth) samples from 14 or 11 of 21 patients, respectively. More importantly, significant levels of peptide-specific IgG were detected in the postvaccination tumor cavity or spinal fluid of all of the tested patients who showed favorable clinical responses. Clinical responses were 5 partial responses, 8 cases of stable disease, and 8 cases of progressive disease. The median overall survival for patients with recurrent glioblastoma multiforme in this study (n = 17) was 622 days. Conclusions: Personalized peptide vaccinations were recommended for the further clinical study to malignant glioma patients.

Edaravone attenuates cerebral ischemic injury by suppressing aquaporin-4

Aquaporin-4 (AQP4) plays a role in the generation of post-ischemic edema. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, epilepsy, traumatic brain injury, and other disorders of the central nervous system (CNS) associated with altered brain water balance. Edaravone, a free radical scavenger, is used for the treatment of acute ischemic stroke (AIS) in Japan. In this study, edaravone significantly reduced the infarct area and improved the neurological deficit scores at 24h after reperfusion in a rat transient focal ischemia model. Furthermore, edaravone markedly reduced AQP4 immunoreactivity and protein levels in the cerebral infarct area. In light of observations that edaravone specifically inhibited AQP4 in a rat transient focal ischemia model, we propose that edaravone might reduce cerebral edema through the inhibition of AQP4 expression following cerebral infarction.

Expression of the SART3 tumor-rejection antigen in brain tumors and induction of cytotoxic T lymphocytes by its peptides.

The authors recently reported on the SART3 tumor-rejection antigen, which possesses epitopes that can induce cytotoxic T lymphocytes (CTLs) in patients with epithelial cancer. To explore a new modality for treatment of patients with brain tumors, this study investigated the expression of the SART3 antigen in patients with brain tumors and the ability of SART3 peptides to induce CTLs from peripheral blood mononuclear cells (PBMCs) of these patients. The SART3 antigen was detected in the cytoplasmic fraction of all 18 glioma cell lines examined and in the majority (31 of 34; 91%) of brain tumor tissues irrespective of their histologies. It was also expressed in the nuclear fraction of all 18 glioma cell lines and in the majority (26 of 34; 76%) of brain tumor tissues. In contrast, the SART3 was not expressed in nontumorous brain tissues. Cytotoxic T lymphocytes were induced in patients with glioma by stimulation with two epitope peptides of SART3. These CTLs could eliminate glioma cells in a HLA-A24–restricted manner. Therefore, the SART3 peptides may be appropriate molecules for use in peptide-based specific immunotherapy of HLA-A24+ patients with brain tumors.

Expression of the tumor-rejection antigen SART1 in brain tumors.

We have reported a tumor‐rejection antigen, SART1259, possessing tumor epitopes capable of inducing cytotoxic T lymphocytes (CTLs) in epithelial‐cancer patients. This study investigated the expression of SART1259 antigen in brain tumors, to explore for a potential molecule for use in specific immunotherapy of patients with brain tumors. The SART1259 antigen was detected in the cytosol fraction of 13 of 18 (72%) glioma cell lines and in 12 of 34 (35%) brain‐tumor tissues, with a higher rate of expression among malignant gliomas (5/10, 50%) and schwannomas (3/4). HLA‐A24‐restricted and SART1‐specific CTLs recognized the HLA‐A24+ and SART1259+ glioma cells, and the levels of recognition correlated both with HLA‐A24‐antigen expression level and with the concentration of the SART1 peptide antigen. Therefore, the SART1259 antigen could be a target molecule for specific immunotherapy of patients with brain tumors expressing HLA‐class‐I antigens. Int. J. Cancer 83:760–764, 1999.

The Free Radical Scavenger Edaravone Rescues Rats from Cerebral Infarction by Attenuating the Release of High-Mobility Group Box-1 in Neuronal Cells

Edaravone, a potent free radical scavenger, is clinically used for the treatment of cerebral infarction in Japan. Here, we examined the effects of edaravone on the dynamics of high-mobility group box-1 (HMGB1), which is a key mediator of ischemic-induced brain damage, during a 48-h postischemia/reperfusion period in rats and in oxygen-glucose-deprived (OGD) PC12 cells. HMGB1 immunoreactivity was observed in both the cytoplasm and the periphery of cells in the cerebral infarction area 2 h after reperfusion. Intravenous administration of 3 and 6 mg/kg edaravone significantly inhibited nuclear translocation and HMGB1 release in the penumbra area and caused a 26.5 ± 10.4 and 43.8 ± 0.5% reduction, respectively, of the total infarct area at 24 h after reperfusion. Moreover, edaravone also decreased plasma HMGB1 levels. In vitro, edaravone dose-dependently (1–10 μM) suppressed OGD- and H2O2-induced HMGB1 release in PC12 cells. Furthermore, edaravone (3–30 μM) blocked HMGB1-triggered apoptosis in PC12 cells. Our findings suggest a novel neuroprotective mechanism for edaravone that abrogates the release of HMGB1.

HLA class-I-restricted and tumor-specific CTL in tumor-infiltrating lymphocytes of patients with gastric cancer

Immune recognition of human cancers except melanoma is not well understood at either the cellular or the molecular level. We demonstrate in this study the existence of HLA class‐I‐restricted and tumor‐specific CTL in IL‐2‐activated TIL (tumor‐infiltrating lymphocytes) of all 4 gastric cancer patients tested. We established HLA A2‐restricted and adenocarcinoma‐specific CTL in 2 HLA A0201+ patients, and HLA A2402‐restricted CTL recognizing both adenocarcinoma and squamous‐cell carcinomas (SCC) in the 2 remaining HLA A2402+ patients. Further, HLA A3101‐restricted and adenocarcinoma‐specific CTL were established in 1 of the 2 HLA A2402+ patients who had HLA A3101 allele. HLA A2‐, A2402‐ and A3101‐restricted CD8+ CTL clones were established from these parental CTL lines. The 2 HLA A2‐restricted CTL lines lysed 8 of 13 HLA A2+ adenocarcinoma cell lines established from different organs (stomach, colon, lung and breast) with different subtypes (HLA A0201, A0206 and A0207). The HLA A2‐restricted CTL line recognized 9 and 6 different HPLC fractions of peptides eluted from the HLA A0201+ breast and HLA A0201+ colon adenocarcinoma cell lines, respectively. Allele‐specific deletion of HLA A2 or A24 molecules was observed in some tumor lines that were not susceptible to lysis by the CTL lines. These results suggest that TIL of gastric cancer possess CTL recognizing different peptide antigens binding to different HLA‐A alleles that are widely expressed on adenocarcinomas and also, to some extent, on SCC from different organs. Int. J. Cancer 70:631–638, 1997.

Minocycline attenuates both OGD-induced HMGB1 release and HMGB1-induced cell death in ischemic neuronal injury in PC12 cells.

High mobility group box-1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic insult and exacerbates brain tissue damage in rats. Minocycline is a semisynthetic second-generation tetracycline antibiotic which has recently been shown to be a promising neuroprotective agent. In this study, we found that minocycline inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated PC12 cells and triggered the activation of p38mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/2). The ERK kinase (MEK)1/2 inhibitor U-0126 and p38MAPK inhibitor SB203580 blocked HMGB1 release in response to OGD. Furthermore, HMGB1 triggered cell death in a dose-dependent fashion. Minocycline significantly rescued HMGB1-induced cell death in a dose-dependent manner. In light of recent observations as well as the good safety profile of minocycline in humans, we propose that minocycline might play a potent neuroprotective role through the inhibition of HMGB1-induced neuronal cell death in cerebral infarction.

Beyond neurological disease: New targets for edaravone (Review)

Free radicals play major roles in the pathogenesis of tissue damage in many diseases and clinical conditions, and the removal of free radicals may offer a treatment option. Several modulators of free radical scavenger pathways have been developed and some have progressed to clinical trials. One such agent, edaravone, was approved in 2001 in Japan for the treatment of cerebral infarction. It has since been shown that edaravone can diffuse into many organs and, in addition to its effects on hydroxyl radical removal, edaravone modulates inflammatory processes, matrix metalloproteinase levels, nitric oxide production, apoptotic cell death, and necrotic cell death. Edaravone also exerts protective effects in a number of animal models of disease and tissue damage, including models of myocardial, lung, intestinal, liver, pancreatic and renal injury. Together with the proven safety of edaravone following 9 years of use as a modulator of free radical scavenging pathways in neurological disease, these additional effects of edaravone suggest that it may offer a novel treatment for several non-neurological diseases and clinical conditions in humans.

Edaravone: a new therapeutic approach for the treatment of acute stroke.

Acute stroke, including acute ischemic stroke (AIS) and acute hemorrhagic stroke, (AHS) is a common medical problem with particular relevance to the demographic changes in industrialized societies. In recent years, treatments for AIS have emerged, including thrombolysis with tissue plasminogen activator (t-PA). Although t-PA is the most effective currently available therapy, it is limited by a narrow therapeutic time window and side effects, and only 3% of all AIS patients receive thrombolysis. Edaravone was originally developed as a potent free radical scavenger and, since 2001, has been widely used to treat AIS in Japan. It was shown that edaravone extended the narrow therapeutic time window of t-PA in rats. The therapeutic time window is very important for the treatment of AIS, and early edaravone treatment is more effective. Thus, more AIS patients might be rescued by administering edaravone with t-PA. Meanwhile, edaravone attenuates AHS-induced brain edema, neurologic deficits and oxidative injury in rats. Although edaravone treatment is currently only indicated for AIS, it does offer neuroprotective effects against AHS in rats. Therefore, we hypothesize that early administration of edaravone can rescue AHS patients as well as AIS patients. Taken together, our findings suggest that edaravone should be immediately administered on suspicion of acute stroke, including AIS and AHS.

Establishment of an enzyme-linked immunosorbent assay (ELISA) for measuring cellular MAGE-4 protein on human cancers

The MAGE genes encoding tumor-rejection antigens are expressed on various human cancers. An enzyme-linked immunosorbent assay (ELISA) was established for measuring cellular MAGE-4 protein (MAGE-4a and/or -4b) expressed on human tumor cells using a monoclonal antibody (mAb) and polyclonal Ab to recombinant MAGE-4b protein. Both the R5 mAb (IgG1) and the polyclonal Ab recognized a 45 kDa protein in extracts of MAGE-4 mRNA positive cancers, and showed no apparent cross-reactivity to the other MAGE gene products (MAGE-1, -2, -3, -6, and -12) by the immunoblot analyses. The R5 mAb and the polyclonal Ab primarily recognized one (the position 119-133) and two oligopeptides (the positions 119-133 and 259-273), respectively, among a series of 31 different MAGE-4b oligopeptides. The amino acid sequences of these two peptides were identical to those of MAGE-4a and -4b, but differed from those of all the other MAGE proteins (MAGE-1, -2, -3, -6, and -12). Substitution of glycine for amino acid in position 123 (arginine, R), 124 (lysine, K), 126 (R) or 128 (K) in a MAGE-4b oligopeptide of the position 119-132 severely decreased the reactivity of the R5 mAb to the oligopeptide. This ELISA also showed no apparent cross-reactivity with the other MAGE gene products (MAGE-1, -2, -3, -6, and -12). The minimum detectable level of MAGE-4 protein was determined to be 10 pg/well (100 pg/ml). The results suggest that this ELISA is a reliable and quantitative method to measure cellular MAGE-4 protein that is a potential target molecule for specific immunotherapy of human cancers.