Ryuji Okuyama

Anti-CD137 Monoclonal Antibody Administration Augments the Antitumor Efficacy of Dendritic Cell-Based Vaccines

In weakly and poorly immunogenic tumor models, we examined the effects of stimulating CD137 (4-1BB) in vivo by administering anti-CD137 monoclonal antibody after tumor lysate-pulsed dendritic cell (TP-DC) vaccination. TP-DC subcutaneous vaccination induced a transient up-regulation of CD137 on T cells and natural killer (NK) cells within vaccine-primed lymph nodes (VPLNs). In established pulmonary and subcutaneous tumor models, anti-CD137 synergistically enhanced tumor regression after TP-DC vaccination. In the subcutaneous tumor model, the combined therapy resulted in improved survival. Combined therapy also resulted in improved local control of subcutaneous tumor after surgical resection. Anti-CD137 polarized the cytokine release of VPLNs and spleen cells in response to tumor antigen toward a type 1 (interferon-γ) versus a type 2 (interleukin-4) profile. Cell depletion and the use of knockout animals identified that CD8+, CD4+, and NK cells were involved in the tumor rejection response and that CD8+ cells had the major effector role. Anti-CD137 administration resulted in increased proliferation of adoptively transferred OT-1 CD8+ T cells in the VPLNs of mice inoculated with B16-OVA TP-DCs. Polarization toward type 1 (interferon-γ) versus type 2 (interleukin-4) was also observed with the OT-1 cells from VPLNs and spleen cells after anti-CD137 injections. This polarization effect was abrogated by the in vivo depletion of NK cells. These findings indicate that the adjuvant effect of anti-CD137 given in conjunction with TP-DC vaccination is associated with the polarization of T effector cells toward a type 1 response to tumor antigen and is mediated via NK cells.

Mechanisms involved in radiation enhancement of intratumoral dendritic cell therapy.

We have previously reported that local tumor irradiation, without inducing cell death, can augment the therapeutic efficacy of intratumoral (IT) dendritic cell (DC) vaccination. This study examined potential mechanisms underlying radiation enhancement of IT DC therapy in this setting. Even though ionizing radiation did not mediate tumor cell killing, bone marrow-derived DCs acquired in vitro tumor antigens from irradiated D5 murine melanoma cells more efficiently than from untreated cells. This radiation-enhanced loading of DCs did not induce DC maturation, but was associated with improved cross-priming of T cells both in vitro and in vivo. Furthermore, in vivo pulsing of DCs with irradiated versus untreated tumor cells resulted in superior presentation of tumor antigens to T cells. In addition, tumor irradiation facilitated homing of IT administered DCs to the draining lymph node, possibly by down-regulating CCL21 expression within the tumor mass. Studies of the tumor microenvironment in irradiated versus untreated tumors did not reveal significant inflammatory changes. Moreover, radiation did not promote accumulation of CD4+ or CD8+ effector T cells within solid tumors. Our results indicate that, without inducing cytotoxicity, tumor irradiation can enhance the ability of DCs to capture tumor antigens, migrate to the draining lymph node, and present processed antigens to T cells. These findings may prove useful in designing future strategies for human cancer immunotherapy.

Immunological responses to a multi-peptide vaccine targeting cancer-testis antigens and VEGFRs in advanced pancreatic cancer patients

The prognosis of patients with advanced pancreatic cancer is extremely poor and there are only a few standard treatments. Here, we report the results of a Phase I clinical trial to investigate the safety, immunostimulatory effects, and antineoplastic activity of a multi-target vaccine composed of four distinct peptides derived from cancer-testis (CT) antigens and vascular endothelial growth factor receptors (VEGFRs). Nine patients with unresectable, advanced pancreatic cancer who were refractory to standard chemotherapy were enrolled. Each patient was vaccinated with HLA-A*2402-restricted peptides derived from the CT antigens kinesin family member 20A (KIF20A) and cell division cycle-associated 1 (CDCA1) as well as from VEGFR1 and VEGFR2 subcutaneously once a week, and disease progression was evaluated up to 6 mo later. Adverse events were assessed using the Common Terminology Criteria for Adverse Events v. 3.0. Immunological responses were monitored by ELISPOT assays and flow cytometry based on peptide-specific dextramers. The clinical outcomes that were measured were tumor response, progression-free survival (PFS) and overall survival (OS). In general, the multi-peptide vaccine was well-tolerated, and no grade 3 or 4 adverse events were observed upon vaccination. Peptide-specific T-cell responses were detected in all 9 patients, and clinical benefits were observed in four of them. Median PFS and OS were 90 and 207 d, respectively. The elicitation of multiple and robust peptide-specific T-cell responses as well as the status of host lymphocytes may be useful prognostic factors among patients with advanced pancreatic cancer treated with peptide-based anticancer vaccines.

Abstract CT124: A phase II study of bevacizumab and irinotecan as second-line therapy for patients with metastatic colorectal cancer previously treated with fluoropyrimidine, oxaliplatin, and bevacizumab

Background: FOLFIRI plus bevacizumab (BV) is widely used as second-line chemotherapy for patients with metastatic colorectal cancer (mCRC) previously treated with FOLFOX/XELOX plus bevacizumab. However, there is insufficient data characterizing the effectiveness of administration of fluoropyrimidine beyond first disease progression. FOLFIRI requires using a CV catheter. Oral fluoropyrimidine may cause severe diarrhea when administered with irinotecan (CPT-11). CPT-11+BV may overcome those problems. Methods: Patients with mCRC previously treated with at least four courses of fluoropyrimidine, oxaliplatin, and bevacizumab were designated to receive 150 mg/m2 of CPT-11 and 10 mg/kg of BV, every 2 weeks as second-line therapy. The primary endpoint was progression-free survival (PFS), and secondary endpoints included response rate (RR), overall survival (OS), and adverse events. Results: Thirty patients from six institutes were enrolled from March 2011 to January 2014. Twenty-five (83.3%) patients were refractory to first-line therapy and eight patients (16.6%) were unable to tolerate first-line therapy. The median PFS was 5.7 months (95% CI; 4.2-7.3 months) and the RR was 6.7% (range, 0.8-22.1%. Grades 3-4 adverse events included leucopenia (36.7%), neutropenia (50%), thrombocytopenia (26.7%), anemia (30%), diarrhea (3.3%), anorexia (6.7%), and hypertension (3.3%). Relative dose intensities were 94.5% and 96.3% for CPT-11 and BV, respectively. Median OS was 11.8 months (6.3 months-not reached). Conclusions: Administration of CPT-11 plus BV to patients with mCRC achieved comparable efficacy with relatively lower toxicities compared with the results of previous studies using FOLFIRI plus BV as second-line therapy. The dose intensity of CPT-11 was judged satisfactory. Clinical trial information: UMIN000005228 Citation Format: Go Nakajima, Hidekazu Kuramochi, Masayuki Ando, Michio Itabashi, Kazuyuki Kawakami, Mie Hamano, Eiichi Hirai, Takayuki Iino, Hajime Yokomizo, Ryuji Okuyama, Tatsuo Araida, Kazuhiko Yoshimatsu, Shingo Kameoka, Kazuhiko Hayashi. A phase II study of bevacizumab and irinotecan as second-line therapy for patients with metastatic colorectal cancer previously treated with fluoropyrimidine, oxaliplatin, and bevacizumab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT124.

Abstract 5038: Non-cording microRNA hsa-miR-199a expression in hepatocellular carcinoma correlates with patient survival

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL [Aim] MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at a post-transcriptional level. Previous study from our group identified forty nine miRNAs that are expressed differentially by comparing 26 pair samples of hepatocellular carcinoma (HCC) tumor tissue and non-tumor liver tissue using next-generation sequencer system. In this study, the expression of hsa-miR-199a and hsa-miR-199b, one of the extracted targets from our study, were investigated by real-time PCR using validation set HCC samples, and were examined the correlation between the miRNA expression and clinical data. [Patients and Methods] Forty-eight patients diagnosed as HCC had undergone surgical removal from 1991 to 1997 at the Department of Surgery, Institute of Gastroenterology, Tokyo Womens Medical University, Japan. The formalin-fixed paraffin embedded samples were prepared using the standard protocol. The paraffin blocks were cut into 10μm sections, and the tumor samples were collected using Laser-captured microdissection and total RNA including miRNA fraction were isolated. After synthesized cDNA using miRNA specific primer, real-time PCR was performed using miRNA specific primer/probe. [Results] The expression level of hsa-miR-199a and hsa-miR-199b were down-regulated in tumor compared to non-tumor (P<0.0001, P=0.0002, respectively). The patients were divided into two groups, higher/lower expression of each miRNA, the group of the higher hsa-miR-199a expression was significantly longer overall survival time compared to the lower expression group (P=0.0303). However, hsa-miR-199b was not significant difference (P=0.3801). [Conclusion] The hsa-miR-199a may be potential prognostic factor for HCC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5038. doi:1538-7445.AM2012-5038

Abstract 5249: Analysis of gene expression levels associating with metabolism of 5-fluorouracil and folate in primary colorectal cancer and corresponding synchronous or metachronous liver metastasis

Background Gene expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) have been shown to be associated with response to 5-fluorouracil-based therapies. Folylpolyglutamate synthase (FPGS), gamma-glutamyl hydrolase (GGH) and dihydrofolate reductase (DHFR) are associated with folate metabolism which regulates intracellular folate level. Analyzing these gene expression levels in liver metastases is important to obtain the prediction of therapy, however, only surgical specimens of the primary tumor are readily available for analysis in many cases. Our aim is to determine how TS, DPD, TP, OPRT, FPGS, GGH and DHFR gene expression levels in primary colorectal cancer (CRC) are related to those in liver metastases. Methods Formalin-fixed, paraffin-embedded tumor specimens from 50 pairs of primary CRC and corresponding liver metastases, including 30 synchronous and 20 metachronous metastases, were dissected by using laser-captured microdissection. Total RNA was extracted and cDNA was prepared by reverse-transcription. Quantitation of target genes and internal reference gene, beta-2-microglobulin, was performed using real-time RT-PCR. Results Significant difference was seen between mRNA expression levels of TS, DPD, FPGS and DHFR in primary tumor and those in corresponding synchronous liver metastases (median value: TS 1.18 vs. 0.05; p Conclusion A good prediction of TP, OPRT and FPGS mRNA levels in synchronous liver metastases as well as DPD and GGH mRNA levels in metachronous liver metastasis can be obtained by measuring those of primary CRC, although no correlation was seen for other genes. We are currently investigating more number of cases and genes related with sensitivity to antitumor drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5249. doi:10.1158/1538-7445.AM2011-5249

Abstract 5585: Optimal conditions for dendritic cells labeling with superparamagnetic iron oxide for cellular magnetic resonance imaging

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Non-invasive cell-tracking in vivo could be an invaluable tool in cell-based therapy, which determines whether or not administrated cells effectively reach their target sites. Recent reports have shown that magnetic resonance imaging (MRI) is a useful and feasible tool to detect in vivo migration and biodistribution of magnetically labeled cells. In this study, we examined optimal conditions for labeling monocyte-derived dendritic cells (Mo-DCs) with a MR contrast agent (Resovist®) consisting of superparamagnetic iron oxide (SPIO) particles. We found that simple addition of Resovist® in the culture medium makes it possible to label Mo-DCs sufficiently and efficiently, and SPIO-uptake appeared to be saturated by its 100μg/ml treatment. FCM analysis showed that expression levels of CD80, CD86, CCR7, HLA-A,B,C and HLA-DR in SPIO-labeling Mo-DCs (100μg/ml Resovist®-treated) were similar to those in non-labeling cells. In vitro migration assays revealed that there were no significant differences between labeling and non-labeling cells in both chemotactic activities to CCL19 and random migration capacities on fibronectin-coated plates. In addition, IFNγ-ELISPOT assay showed labeling and non-labeling Mo-DCs were able to stimulate antigen-specific T cells to a similar extent, indicating that Resovist®-treatment did not affect the capacities of Mo-DCs to take up, process and present antigens to T cells. Taken together, we concluded that labeling of Mo-DCs with SPIO, particularly with 100μg/ml Resovist® could be a potent method for monitoring trafficking and biodistribution of these cells in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5585.

Abstract 2544: Analysis of the mechanism for acquiring resistance to 5-FU in gastric cancer cell lines

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background 5-Fluorouracil (5-FU) still remains the key drug for the treatment of gastric cancer. Thus, analysis of the mechanism for acquiring resistance of gastric cancer cells to 5-FU and the development of effective chemotherapy for 5-FU-resistant gastric cancer are important. Methods From the parent human gastric cancer cell lines (MKN45, MKN74, KATOIII and NCI-N87), we established several 5-FU-resistant cell lines (MKN45/5FU, MKN74/5FU, KATOIII/5FU and NCI-N87/5FU) by continuous exposure of the cells to progressively increasing concentrations of 5-FU over a year. We then compared the sensitivity of the parent cell lines to 5-FU-resistant cell lines by MTT assay. We also quantitatively evaluated the mRNA expression levels of four genes known to be associated with 5-FU metabolism, namely, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyltransferase (OPRT), by real-time RT-PCR assay. Results The IC50 ratio of each 5-FU resistant cell line to its parent cell line was as follows; MKN45/5FU: 10.0; MKN74/5FU: 5.5; KATOIII/5FU: 2.2; NCI-N87/5FU: 4.8. Comparing with the corresponding parent cell lines, the 5-FU resistant cell lines, MKN45/5FU, MKN74/5FU, KATOIII/5FU and NCI-N87/5FU, showed a 0.62, 0.40, 0.37 and 0.56-fold decrease in TS gene expression, respectively. In addition, three of the four resistant cell lines showed a tendency towards increase in the gene expression level of DPD and decrease in that of TP. No significant changes in the expression level of the gene encoding OPRT was noted in any of the 5-FU-resistant cell lines. Conclusion We established four 5-FU-resistant lines from the corresponding parent gastric cancer cell lines and demonstrated the various changes in the expression profiles of the genes known to be associated with 5-FU metabolism. These results suggest that the mechanism of acquiring resistance to 5-FU may vary among gastric tumors. To identify the genetic characteristics of these resistant cell lines, we propose to analyze the gene expression profiles at several time-points from the beginning of establishment of 5-FU-resistance, using microarray-based technology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2544.