Evaluation of Toxic Amyloid 42 Oligomers in Rat Primary Cerebral Cortex Cells and Human iPS-derived Neurons Treated with 10-Me-Aplog-1, a New PKC Activator.

Amyloid ß42 (Aß42), a causative agent of Alzheimer's disease (AD), is derived extracellularly from Aß precursor protein (APP) following the latter's cleavage by ß-secretase, but not α-secretase. Protein kinase Cα (PKCα) activation is known to increase α-secretase activity, thereby suppressing Aß production. Since Aß42 oligomer formation causes potent neurotoxicity, APP modulation by PKC ligands is a promising strategy for AD treatment. Although bryostatin-1 (bryo-1) is a leading compound for this strategy, its limited natural availability and the difficulty of its total synthesis impedes further research. To address this limitation, Irie and colleagues have developed a new PKC activator with few side effects, 10-Me-Aplog-1, (1), which decreased Aß42 in the conditioned medium of rat primary cerebral cortex cells. These results are associated with increased α-secretase but not PKCε-dependent Aß-degrading enzyme. The amount of neuronal embryonic lethal abnormal vision (nELAV), a known ß-secretase stabilizer, was reduced by treatment with 1. Notably, 1 prevented the formation of intracellular toxic oligomers. Furthermore, 1 suppressed toxic oligomerization within human iPS-derived neurons such as bryo-1. Given that 1 was not neurotoxic toward either cell line, these findings suggest that 1 is a potential drug lead for AD therapy.

Perioperative plasma glypican-3 level may enable prediction of the risk of recurrence after surgery in patients with stage I hepatocellular carcinoma.

Glypican-3 (GPC3) is a glycosylphosphatidylinositol-anchored cell surface protein overexpressed in hepatocellular carcinoma(HCC), and its overexpression is associated with poor prognosis. The diagnostic potential of GPC3 as a serum marker has been reported. In the present study, we evaluated the usefulness of plasma GPC3 as a predictor for recurrence after surgical resection in stage I HCC patients by newly developed an enzyme-linked immunosorbent assay (ELISA) system. Current study demonstrated that high levels of preoperative plasma GPC3 patients tended to experience postoperative recurrence. On the other hand, pre- and postoperative plasma GPC3 positivity of non-recurrence patients was very low. Moreover, even after surgery, approximately half of patients who experienced recurrence were positive for plasma GPC3. Postoperative plasma GPC3 positivity was significantly correlated with worse recurrence-free survival. Immuohistochemical analysis also showed positive rate of GPC3-expression in HCC was higher in recurrence patients than in non-recurrence patients. These results suggested that both pre- and postoperative plasma GPC3 levels may be accurate predictors for recurrence after curative resection of early-stage HCC. It should be noted that the current study only examined a small number of cases; thus, a larger sample size is necessary to validate GPC3 as a predictor for HCC recurrence.

Phase II study of the GPC3-derived peptide vaccine as an adjuvant therapy for hepatocellular carcinoma patients.

The recurrence rates of Hepatocellular carcinoma (HCC) are high, necessitating novel and effective adjuvant therapies. Therefore, we conducted a phase II study of glypican-3 (GPC3) peptide vaccine as an adjuvant therapy for HCC patients. Forty-one patients with initial HCC who had undergone surgery or radiofrequency ablation (RFA) were analyzed in this phase II, open-label, single-arm trial. Ten vaccinations were performed for 1 y after curative treatment. We also investigated case-control subjects, where selected patients treated surgically during the same period were analyzed. The expression of GPC3 in the available primary tumors was determined by immunohistochemical analysis. Six patients received RFA therapy while 35 received surgery. The recurrence rate tended to be lower in the 35 patients treated with surgery plus vaccination compared to 33 patients who underwent surgery alone (28.6% vs. 54.3% and 39.4% vs. 54.5% at 1 and 2 y, respectively; p = 0.346, 0.983). Twenty-five patients treated with surgery and vaccination had GPC3-positive tumors; the recurrence rate in this group was significantly lower compared to that in 21 GPC3-positive patients who received surgery only (24% vs. 48% and 52.4% vs. 61.9% at 1 and 2 y, respectively; p = 0.047, 0.387). The GPC3 peptide vaccine improved the 1-y recurrence rate in patients with GPC3-positive tumors. This study demonstrated that GPC3 expression by the primary tumor may be used as a biomarker in a putative larger randomized clinical trial to determine the efficacy of the GPC3-derived peptide vaccine.

A peptide antigen derived from EGFR T790M is immunogenic in non­small cell lung cancer.

Lung cancer is the leading cause of cancer­related deaths worldwide. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, have demonstrated marked clinical activity against non-small cell lung cancer (NSCLC) harboring activating epidermal growth factor receptor (EGFR) mutations. However, in most cases, patients develop acquired resistance to EGFR­TKI therapy. The threonine to methionine change at codon 790 of EGFR (EGFR T790M) mutation is the most common acquired resistance mutation, and is present in ~50% cases of TKI resistance. New treatment strategies for NSCLC patients harboring the EGFR T790M mutation are required. We evaluated the immunogenicity of an antigen derived from EGFR with the T790M mutation. Using BIMAS we selected several EGFR T790M­derived peptides bound to human leukocyte antigen (HLA)-A*02:01. T790M-A peptide (789-797) (IMQLMPFGC)-specific cytotoxic T lymphocytes (CTLs) were induced from peripheral blood mononuclear cells (PBMCs) of HLA-A2+ healthy donors. An established T790M-A-specific CTL line showed reactivity against the NCSLC cell line, H1975-A2 (HLA-A2+, T790M+), but not H1975 (HLA-A2-, T790M+), and the corresponding wild-type peptide (ITQLMPFGC)-pulsed T2 cells using an interferon-γ (IFN-γ) enzyme-linked immuno spot (ELISPOT) assay. This CTL line also demonstrated peptide-specific cytotoxicity against H1975-A2 cells. This finding suggests that the EGFR T790M mutation-derived antigen could be a new target for cancer immunotherapy.

Identification of a novel HLA-A 02:01-restricted cytotoxic T lymphocyte epitope derived from the EML4-ALK fusion gene.

Cancer immunotherapy is a promising new approach to cancer treatment. It has been demonstrated that a high number of tumor-specific cytotoxic T cells (CTLs) is associated with increased disease-specific survival in lung cancer patients. Identification of superior CTL epitopes from tumor antigens is essential for the development of immunotherapy for malignant tumors. The EML4-ALK fusion gene was recently identified in a subset of non-small cell lung cancers (NSCLCs). In this study we searched for HLA-A 02:01- and HLA-A 24:02­restricted epitopes derived from EML4-ALK by screening predicted EML4-ALK­derived candidate peptides for the induction of tumor­reactive CTLs. Nine EML4-ALK­derived peptides were selected by a computer algorithm based on a permissive HLA-A 02:01 or HLA-A 24:02 binding motif. One of the nine peptides induced peptide-specific CTLs from human peripheral blood mononuclear cells. We were able to generate a peptide­specific CTL clone. This CTL clone specifically recognized peptide­pulsed T2 cells and H2228 cells expressing HLA-A 02:01 and EML4-ALK that had been treated with IFN-γ 48 h prior to examination. CTL activity was inhibited by an anti-HLA­class I monoclonal antibody (W6/32), consistent with a class I-restricted mechanism of cytotoxicity. These results suggest that this peptide (RLSALESRV) is a novel HLA-A 02:01-restricted CTL epitope and that it may be a new target for antigen-specific immunotherapy against EML4­ALK-positive cancers.