YKL-40 downregulation is a key factor to overcome temozolomide resistance in a glioblastoma cell line.

The frequent recurrence of glioblastoma multiforme (GBM) after standard treatment with temozolomide (TMZ) is a crucial issue to be solved in the clinical field. O6­methylguanine­DNA methyltransferase (MGMT) is considered one of the major mechanisms involved in TMZ resistance. However, some important mechanisms for TMZ resistance other than MGMT have recently been identified. In the present study, we established a TMZ-resistant (TMZ-R) U87 glioblastoma cell line in vitro and in vivo and investigated novel targeting molecules other than MGMT in those cells. The TMZ-R U87 glioblastoma cell line was established in vitro and in vivo. TMZ-R U87 cells showed a more invasive activity and a shorter survival time in vivo. Gene expression analysis using DNA microarray and quantitative PCR (qPCR) demonstrated that YKL­40, MAGEC1 and MGMT mRNA expression was upregulated 100-, 83- and 6-fold, respectively in the TMZ-R U87 cell line. Western blot analysis and qPCR demonstrated that STAT3 phosphorylation, STAT3 target genes and stem cell and mesenchymal marker genes were upregulated to a greater extent in the TMZ­resistant cell line. Notably, short hairpin (sh)RNA­based inhibition against the YKL­40 gene resulted in moderate growth inhibition in the resistant cells in vitro and in vivo. Additionally, YKL­40 gene inhibition exhibited significant suppression of the invasive activity and particularly partially restored the sensitivity to TMZ. Therefore, YKL­40 may be a novel key molecule in addition to MGMT, that is responsible for TMZ resistance in glioblastoma cell lines and could be a new target to overcome TMZ resistance in recurrent glioblastomas in the future.

The identification of affinity peptide ligands specific to the variable region of human antibodies.

Of all potential biological therapeutics, monoclonal antibody (mAb)-based therapies are becoming the dominant focus of clinical research. In particular, smaller recombinant antibody fragments such as single-chain variable fragments (scFv) have become the subject of intense focus. However, an efficient affinity ligand for antibody fragment purification has not been developed. In the present study, we designed a consensus sequence for the human antibody heavy or light chain-variable regions (Fv) based on the antibody sequences available in the ImMunoGeneTics information system (IMGT), and synthesized these consensus sequences as template Fv antibodies. We then screened peptide ligands that specifically bind to the repertoire-derived human Fv consensus antibody using a 12-mer-peptide library expressed-phage display method. Subsequently, 1 peptide for the VH template and 8 peptides for the VK template were selected as the candidate ligands after 4 rounds of panning the phage display. Using peptide-bead-based immunoprecipitation, the code-4 and code-13 peptides showed recovery rates of the VH and VK templates that were 20-30% and 40-50%, respectively. Both peptides exhibited better recovery rates for trastuzumab scFv (approximately 40%). If it were possible to identify the best combination of VH and VK-binding peptides among the ligand peptides suitable for the human mAb Fv sequence, the result could be a promising purification tool that might greatly improve the cost efficiencies of the purification process.

Anti-vascular endothelial growth factor receptor (VEGFR) 2 autoantibody identification in glioblastoma patient using single B cell-based antibody gene cloning.

Antibody direct cloning from single B cells is simple and efficient and has been successful in antibody identification of infectious diseases. However, although a recent whole-exome sequencing revealed abundant heterogeneic mutation accumulation in cancers, identification and synthesis of autoantibodies against specific cancer-associated antigens is still difficult in cancer patients owing to the very small number of B cells producing autoantibodies. In the present study, to identify autoantibodies targeting tumor antigens, we measured the titer of autoantibodies in high-grade glioma patients' plasma and identified two patients with elevated autoantibodies to a few transmembrane proteins. Specific B cells producing autoantibody against vascular endothelial growth factor receptor (VEGFR) 2 were immunostained with labeled protein and anti-human IgG antibody, and then collected by a single cell sorter. Finally, 22 antibody genes were successfully identified using direct IgG cloning from single B cell mRNA, and two antibody clones were found to have significant VEGFR2-specific binding affinity. The current direct human IgG gene cloning technique for identifying human antibodies derived from IgG-memory B cells avoids time-consuming procedures such as phage display-based antibody-library screening, and therefore may be applicable to identifying human autoantibodies in a variety of disorders including cancers even when antibody elevation is not detected because of a very small number of memory B cells.

Novel cancer-testis antigen expression on glioma cell lines derived from high-grade glioma patients.

Glioblastoma multiforme (GBM) is one of the most malignant and aggressive tumors, and has a very poor prognosis with a mean survival time of <2 years, despite intensive treatment using chemo-radiation. Therefore, novel therapeutic approaches including immunotherapy have been developed against GBM. For the purpose of identifying novel target antigens contributing to GBM treatment, we developed 17 primary glioma cell lines derived from high-grade glioma patients, and analyzed the expression of various tumor antigens and glioma-associated markers using a quantitative PCR and immunohistochemistry (IHC). A quantitative PCR using 54 cancer-testis (CT) antigen-specific primers showed that 36 CT antigens were positive in at least 1 of 17 serum-derived cell lines, and 17 antigens were positive in >50% cell lines. Impressively, 6 genes (BAGE, MAGE-A12, CASC5, CTAGE1, DDX43 and IL-13RA2) were detected in all cell lines. The expression of other 13 glioma-associated antigens than CT genes were also investigated, and 10 genes were detected in >70% cell lines. The expression of CT antigen and glioma-associated antigen genes with a high frequency were also verified in IHC analysis. Moreover, a relationship of antigen gene expressions with a high frequency to overall survival was investigated using the Repository of Molecular Brain Neoplasia Data (REMBRANDT) database of the National Cancer Institute, and expression of 6 genes including IL-13RA2 was inversely correlated to overall survival time. Furthermore, 4 genes including DDX43, TDRD1, HER2 and gp100 were identified as MGMT-relevant factors. In the present study, several CT antigen including novel genes were detected in high-grade glioma primary cell lines, which might contribute to developing novel immunotherapy and glioma-specific biomarkers in future.

Effect of the STAT3 inhibitor STX-0119 on the proliferation of cancer stem-like cells derived from recurrent glioblastoma.

Signal transducer and activator of transcription (STAT) 3, a member of a family of DNA-binding molecules, is a potential target in the treatment of cancer. The highly phosphorylated STAT3 in cancer cells contributes to numerous physiological and oncogenic signaling pathways. Furthermore, a significant association between STAT3 signaling and glioblastoma multiforme stem-like cell (GBM-SC) development and maintenance has been demonstrated in recent studies. Previously, we reported a novel small molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. In the present study, we focused on cancer stem-like cells derived from recurrent GBM patients and investigated the efficacy of STX-0119. Three GBM stem cell lines showed many stem cell markers such as CD133, EGFR, Nanog, Olig2, nestin and Yamanaka factors (c-myc, KLF4, Oct3/4 and SOX2) compared with parental cell lines. These cell lines also formed tumors in vivo and had similar histological to surgically resected tumors. STAT3 phosphorylation was activated more in the GBM-SC lines than serum-derived GB cell lines. The growth inhibitory effect of STX-0119 on GBM-SCs was moderate (IC50 15-44 µM) and stronger compared to that of WP1066 in two cell lines. On the other hand, the effect of temozolomide was weak in all the cell lines (IC50 53-226 µM). Notably, STX-0119 demonstrated strong inhibition of the expression of STAT3 target genes (c-myc, survivin, cyclin D1, HIF-1α and VEGF) and stem cell-associated genes (CD44, Nanog, nestin and CD133) as well as the induction of apoptosis in one stem-like cell line. Interestingly, VEGFR2 mRNA was also remarkably inhibited by STX-0119. In a model using transplantable stem-like cell lines in vivo GB-SCC010 and 026, STX-0119 inhibited the growth of GBM-SCs at 80 mg/kg. STX-0119, an inhibitor of STAT3, may serve as a novel therapeutic compound against GBM-SCs even in temozolomide-resistant GBM patients and has the potential for GBM-SC-specific therapeutics in combination with temozolomide plus radiation therapy.

α-type-1 polarized dendritic cell-based vaccination in recurrent high-grade glioma: a phase I clinical trial.

BACKGROUND: High-grade gliomas including glioblastoma multiforme (GBM) are among the most malignant and aggressive of tumors, and have a very poor prognosis despite a temozolomide-based intensive treatment. Therefore, a novel therapeutic approach to controlling recurrence is needed. In the present study, we investigated the effect of activated dendritic cell (DC) (α-type-1 polarized DC)-based immunotherapy on high-grade glioma patients with the HLA-A2 or A24 genotype. METHODS: Nine patients with recurrent high-grade gliomas including 7 with GBMs who fulfilled eligibility criteria were enrolled into a phase I study of monocyte-derived DC-based immunotherapy. HLA-genotyping revealed 1 case of HLA-A*0201 and 8 cases of A*2402. Enriched monocytes obtained using OptiPrep(TM) from leukapheresis products on day1, were incubated with GM-CSF and IL-4 in a closed serum-free system, and activated on day6 with TNF-α, IL-1ß, IFN-α, IFN-γ, and poly I/C. After pulsing with a cocktail of 5 synthetic peptides (WT-1, HER2, MAGE-A3, and MAGE-A1 or gp100) restricted to HLA-A2 or A24 and KLH, cells were cryopreserved until used. Thawed DCs were injected intradermally in the posterior neck at a dose per cohort of 1.0, 2.0 and 5.0× 10(7)/body. RESULTS: The frequency of CD14(+) monocytes increased to 44.6% from 11.9% after gradient centrifugation. After a 7-day-incubation with cytokines, the mean percentage of DCs rated as lin(-)HLA-DR(+) in patients was 56.2 ± 19.1%. Most DCs expressed high levels of maturation markers, co-stimulatory molecules and type-1 phenotype (CD11c+HLA-DR+) with a DC1/2 ratio of 35.6. The amount of IL-12 produced from activated DCs was 1025 ± 443 pg/ml per 10(5) cells. All 76 DC injections were well tolerated except for transient liver dysfunction with grade II. Six patients showed positive immunological responses to peptides in an ELISPOT assay, and positive skin tests to peptide-pulsed DC and KLH were recognized in 4 cases. The clinical response to DC injections was as follows :1 SD and 8 PD. Interestingly, the SD patient, given 24 DC injections, showed a long-term recurrence-free and immunological positive response period. CONCLUSIONS: These results indicate peptide cocktail-treated activated α-type-1 DC-based immunotherapy to be a potential therapeutic tool against recurrent high-grade glioma with mainly HLA-A*2402. TRIAL REGISTRATION: Current non-randomized investigational trial UMIN-CTR UMIN ID: 000000914.

Dendritic cell-based vaccination in metastatic melanoma patients: phase II clinical trial.

Metastatic and chemoresistant melanoma can be a good target of immunotherapy because it is an intractable cancer with a very poor prognosis. Previously, we tested a dendritic cell (DC)-based phase I vaccine, and confirmed that it was safe. In the present study, we performed a phase II trial of a DC vaccine for metastatic melanoma patients with mainly the HLA-A24 genotype, and investigated the efficacy of the vaccine. Twenty-four patients with metastatic melanoma were enrolled into a phase II study of DC-based immunotherapy. The group included 19 HLA-A24-positive (A*2402) patients and 3 HLA-A2-positive (A*0201) patients. The protocol for DC production was similar to that in the phase I trial. Briefly, a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-A2, MAGE-A3 and MART-1 or MAGE­A1) restricted to HLA-A2 or A24 and KLH were used for DC pulsing. Finally, DCs were injected subcutaneously (s.c.) into the inguinal region in the dose range of 1-5x107 per shot. The DC ratio (lin-HLA-DR+) of the vaccine was 38.1±13.3% and the frequency of CD83+ DCs was 25.7±20.8%. Other parameters regarding DC processing were not different from phase I. Immune response-related parameters including the ELISPOT assay, DTH reaction to peptide or KLH, DC injection numbers were shown to be related to a good prognosis. The ELISPOT reaction was positive in 75% of the patients vaccinated. The increase of anti-melanoma antigen antibody titer before vaccination was also shown to be a prognosis factor, but that post-vaccination was not. Based on immunohistochemical analysis, CD8 and IL-17 were not involved in the prognosis. Adverse effects of more than grade III were not seen. Overall survival analysis revealed a significant survival prolongation effect in DC-given melanoma patients. These results suggest that peptide cocktail-treated DC vaccines may be a safe and effective therapy against metastatic melanoma in terms of prolongation of overall survival time.

Identification of novel MAGE-A6- and MAGE-A12-derived HLA-A24-restricted cytotoxic T lymphocyte epitopes using an in silico peptide-docking assay.

Many cancer-testis antigen genes have been identified; however, few human leukocyte antigen (HLA)-A24-restricted cytotoxic T cell (CTL) epitope peptides are available for clinical immunotherapy. To solve this problem, novel tools increasing the efficacy and accuracy of CTL epitope detection are needed. In the present study, we utilized a highly active dendritic cell (DC)-culture method and an in silico HLA-A24 peptide-docking simulation assay to identify novel CTL epitopes from MAGE-A6 and MAGE-A12 antigens. The highly active DCs, called α-type-1 DCs, were prepared using a combination of maturation reagents to produce a large amount of interleukin-12. Meanwhile, our HLA-A24 peptide-docking simulation assay was previously demonstrated to have an obvious advantage of accuracy over the conventional prediction tool, bioinformatics and molecular analysis section. For CTL induction assays, peripheral blood mononuclear cells derived from six cases of HLA-A24(+) melanoma were used. Through CTL induction against melanoma cell lines and peptide-docking simulation assays, two peptides (IFGDPKKLL from MAGE-A6 and IFSKASEYL from MAGE-A12) were identified as novel CTL epitope candidates. Finally, we verified that the combination of the highly active DC-culture method and HLA-A24 peptide-docking simulation assay might be tools for predicting CTL epitopes against cancer antigens.

Antitumor activity of a novel small molecule STAT3 inhibitor against a human lymphoma cell line with high STAT3 activation.

Signal transducer and activator of transcription (STAT)3, a member of a family of DNA-binding molecules mediating numerous physiological and oncogenic signaling pathways, is a novel target in cancer cells which show high phosphorylation of STAT3. Recently, we identified a novel small-molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. We investigated the mechanisms responsible for the antitumor activity in vitro and in vivo through numerous biochemical and biological assays. Specifically, the effects of STX-0119 on target genes (c-myc, cyclin D1, survivin) and apoptosis induction were analyzed in tumors treated with STX-0119 in vivo. STX-0119 showed strong growth-inhibitory activity against a broad range of hematological cancer cell lines, particularly lymphomas. STX-0119 suppressed the growth of SCC3 cells, a human lymphoma cell line with highly activated STAT3, through apoptosis and down-regulation of STAT3 targets such as c-myc, cyclin D1, survivin and Bcl-xL. Notably, Tyr-705-phosphorylated STAT3 up-regulation was not significantly suppressed by STX-0119, as opposed to other STAT3 inhibitors. STX-0119 demonstrated potent antitumor effects in vivo in SCC3-bearing nude mice by way of the down-regulation of STAT3 target genes and induction of apoptosis in the tumors. Thus, STX-0119 may be a new type of STAT3 inhibitor exhibiting strong antitumor activity.

Analysis of HLA-A24-restricted peptides of carcinoembryonic antigen using a novel structure-based peptide-HLA docking algorithm.

Carcinoembryonic antigen (CEA) is a very common tumor marker because many types of solid cancer usually produce a variety of CEA and a highly sensitive measuring kit has been developed. However, immunological responses associated with CEA have not been fully characterized, and specifically a weak immunogenicity of CEA protein as a tumor antigen is reported in human leukocyte antigen (HLA)-A24-restricted CEA peptide-based cancer immunotherapy. These observations demonstrated that immunogenic and potent HLA-A24-restricted CTL epitope peptides derived from CEA protein are seemingly difficult to predict using a conventional bioinformatics approach based on primary amino acid sequence. In the present study, we developed an in silico docking simulation assay system of binding affinity between HLA-A24 protein and A24-restricted peptides using two software packages, AutoDock and MODELLER, and a crystal structure of HLA-A24 protein obtained from the Protein Data Bank. We compared the current assay system with HLA-peptide binding predictions of the bioinformatics and molecular analysis section (BIMAS) in terms of the prediction capability using MHC stabilization and peptide-stimulated CTL induction assays for CEA and other HLA-A24 peptides. The MHC stabilization score was inversely correlated with the affinity calculated in the docking simulation alone (r = -0.589, P = 0.015), not with BIMAS score or the IFN-γ production index. On the other hand, BIMAS was not significantly correlated with any other parameters. These results suggested that our in silico assay system has potential advantages in efficiency of epitope prediction over BIMAS and ease of use for bioinformaticians.

Identification of cytomegalovirus (CMV)pp65 antigen-specific human monoclonal antibodies using single B cell-based antibody gene cloning from melanoma patients.

Recently, because of highly advanced protein engineering technology, beyond the chimeric antibody, highly humanized and fully human antibody development is becoming crucial in the medical field. In the last decade, investigational approaches using clinical samples for fully human antibody production have been performed, but there are still problems with efficiency and accuracy, which should be solved. In the present study, based on novel IgG antibody-measuring ELISA and antibody gene copy number-quantitative PCR, a human single B cell RT-PCR-mediated IgG monoclonal antibody (mAb) gene cloning method was established, and CMVpp65-specific human mAbs were successfully identified. Quantitative PCR for the human IgG mRNA copy number per cell demonstrated that the detection range was 10-250copies/cell. CMVpp65(+)surfaceIgG(+) B cells were collected from melanoma patients who showed high titers of serum anti-CMVpp65 IgG antibody. RT-PCR was successful in 64% (IGH) and 84% (ß-actin) of 88 single B cells. Finally, both IGH and IGL gene amplifications in the same cell were successful in 21 single cells, and 18 IgG antibody genes specific for CMVpp65 antigen were cloned. Four of 13 recombinant human single-chain fragment variable (scFv) antibodies showed strong responses to full-length CMVpp65 protein. These results suggested that the current fully human mAb production procedure through antibody-titer screening by ELISA, single B cell RT-PCR-based antibody gene cloning, and the making of scFv recombinant antibody is an efficient method of therapeutic antibody development.

Identification of a New Series of STAT3 Inhibitors by Virtual Screening.

The signal transducer and activator of transcription 3 (STAT3) is considered to be an attractive therapeutic target for oncology drug development. We identified a N-[2-(1,3,4-oxadiazolyl)]-4-quinolinecarboxamide derivative, STX-0119, as a novel STAT3 dimerization inhibitor by a virtual screen using a customized version of the DOCK4 program with the crystal structure of STAT3. In addition, we used in vitro cell-based assays such as the luciferase reporter gene assay and the fluorescence resonance energy transfer-based STAT3 dimerization assay. STX-0119 selectively abrogated the DNA binding activity of STAT3 and suppressed the expression of STAT3-regulated oncoproteins such as c-myc and survivin in cancer cells. In contrast, a truncated inactive analogue, STX-0872, did not exhibit those activities. Oral administration of STX-0119 effectively abrogated the growth of human lymphoma cells in a SCC-3 subcutaneous xenograft model without visible toxicity. Structure-activity relationships of STX-0119 derivatives were investigated using the docking model of the STAT3-SH2 domain/STX-0119.