Cancer ScienceVolume 104, Issue 1 p. 98-104 Original Article Open Access Phase I trial of OTS11101, an anti-angiogenic vaccine targeting vascular endothelial growth factor receptor 1 in solid tumor Hidetoshi Hayashi, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorTakayasu Kurata, Corresponding Author Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan To whom correspondence should be addressed. E-mail: t-kurata@med.kindai.ac.jpSearch for more papers by this authorYasuhito Fujisaka, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorHisato Kawakami, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorKaoru Tanaka, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorTakafumi Okabe, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorMasayuki Takeda, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorTaroh Satoh, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorKoji Yoshida, Onco Therapy Science, Kanagawa Science Park R&D, Takatsu-ku, Kawasaki City, Kanagawa, JapanSearch for more papers by this authorTakuya Tsunoda, Onco Therapy Science, Kanagawa Science Park R&D, Takatsu-ku, Kawasaki City, Kanagawa, JapanSearch for more papers by this authorTokuzo Arao, Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorKazuto Nishio, Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorKazuhiko Nakagawa, Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this author Hidetoshi Hayashi, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorTakayasu Kurata, Corresponding Author Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan To whom correspondence should be addressed. E-mail: t-kurata@med.kindai.ac.jpSearch for more papers by this authorYasuhito Fujisaka, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorHisato Kawakami, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorKaoru Tanaka, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorTakafumi Okabe, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorMasayuki Takeda, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorTaroh Satoh, Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorKoji Yoshida, Onco Therapy Science, Kanagawa Science Park R&D, Takatsu-ku, Kawasaki City, Kanagawa, JapanSearch for more papers by this authorTakuya Tsunoda, Onco Therapy Science, Kanagawa Science Park R&D, Takatsu-ku, Kawasaki City, Kanagawa, JapanSearch for more papers by this authorTokuzo Arao, Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorKazuto Nishio, Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this authorKazuhiko Nakagawa, Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, JapanSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract OTS11101 is a novel peptide vaccine that acts as an angiogenesis inhibitor by inducing cytotoxic T lymphocyte (CTL) cells that specifically target vascular endothelial cells expressing vascular endothelial growth factor (VEGF) receptor 1. We conducted a phase I study to evaluate the safety, tolerability, maximum tolerated dose, and pharmacodynamic biomarker status of this vaccine. Nine patients with advanced solid tumors received 1.0, 2.0, or 3.0mg of OTS11101 subcutaneously, once a week in a 28-day cycle. Three patients experienced grade 1 injection site reactions, which were the most frequent adverse events. Grade 2 proteinuria and hypertension each occurred in one patient. As other toxicities were generally mild, the maximum tolerated dose was not reached. Furthermore, we explored the induction of specific activated CTLs, and biomarkers related to angiogenesis. A pharmacodynamics study revealed that induction of specific CTLs was observed for a dose of 2.0 and 3.0mg. The serum concentrations of soluble VEGF receptor 1 and 2 after vaccination increased significantly compared with baseline. A microarray was performed to give a comprehensive analysis of gene expression, suggesting that OTS11101 vaccination resulted in T cell activation in a clinical setting. In conclusion, OTS11101 was well tolerated in patients up to 3.0mg once weekly and our biomarker analysis suggested that this anti-angiogenesis vaccine is biologically active. (Cancer Sci 2013; 104: 98–104) Angiogenesis, defined as the formation of new blood vessels from a pre-existing vasculature, is essential for tumor growth and the spread of metastases.1, 2 Therefore, angiogenesis inhibition is considered to be an effective strategy to treat cancer, and clinical application of this strategy is pursued using multiple modalities, which include specific inhibitors of the signaling pathways of vascular endothelial growth factor (VEGF), including their corresponding receptors (VEGFR). The approval of a new broad family of molecularly targeted anticancer drugs, such as anti-VEGF antibody and VEGF receptor tyrosine kinase receptor inhibitors (VEGFR-TKIs), represents one of the most significant recent advances in clinical oncology. However, these existing anti-angiogenic agents have significant shortcomings, including side-effects and the requirement of frequent or continuous administration. To overcome these deficits, novel alternative therapies with different mechanisms of action would be of great value for anti-cancer therapy. Vascular endothelial growth factor receptor 2 is expressed on endothelial cells of vessels in various types of primary tumor and metastases. This receptor has therefore been a major target to date.3 However, it has been emphasized that tumor angiogenesis mediated by the VEGFR1 pathway is also important.4, 5 Previous studies have demonstrated that VEGFR1, but not VEGFR2, is upregulated by hypoxic conditions,6, 7 and similar patterns of distribution of both VEGFR1 and VEGFR2 are not always observed in tumors.8-10 Thus, VEGFR1 is a promising target for anti-angiogenic cancer treatments, and further clinical development of this strategy is warranted. Recently, several specific immunotherapies have been attempted and some clinical trials have shown promising efficacy. In particular, epitope peptides that have been used for the induction of CTL responses in patients with cancer in numerous clinical studies.11 We have previously identified epitope peptides of human VEGFR1 and demonstrated that CTLs induced by these peptides have a potent and specific cytotoxicity, in a human leukocyte antigen (HLA) class I-restricted manner, against not only peptide-pulsed target cells but also endothelial cells endogenously expressing VEGFR1.12 Furthermore, vaccination using these epitope peptides in vivo was shown to inhibit tumor-induced angiogenesis, resulting in significant suppression of tumor growth without fatal adverse effects.12 OTS11101 (VEGFR1-1084) is a novel peptide vaccine restricted with HLA-A*24:02, which acts as an angiogenesis inhibitor by specifically targeting VEGFR1. Here, we conducted a phase I dose-escalation study to determine the maximum tolerated dose (MTD), tolerability, and antitumor effect of OTS11101 administered by a weekly subcutaneous injection. To identify biomarkers that reflect the pharmacodynamics and doseresponse relationship of OTS11101, we further evaluated the CTL response induced with OTS11101 and the serum concentration of soluble VEGFR1, soluble VEGFR2, and various types of cytokines related to angiogenesis. Comprehensive gene expression analysis was performed using a microarray. HLA-A*24:02-positive individuals aged ≥20 years with a histologically confirmed diagnosis of an advanced tumor refractory to standard therapy were included in the study. Within 21days of study registration, patients had to have an absolute white blood cell count of ≥3000 or ≤12000, platelets ≥100000/mm3, and hemoglobin ≥9.0g/dL. Baseline creatinine and total bilirubin had to be less than 1.5 times the upper limit of normal (ULN) and both aspartate aminotransferase (AST) and alanine aminotransferase (ALT) had to be 2.5 times the ULN. Other inclusion criteria included an Eastern Cooperative Oncology Group performance status of 2 and adequate organ function. Individuals were excluded if they had a symptomatic brain tumor or brain metastases, active bleeding, or serious illness or concomitant nononcologic disease that was difficult to control by medication. All subjects received information about the nature and purpose of the study, and they provided written informed consent in accordance with institutional guidelines. This study was designed as a single-center, open-label, dose-escalation phase I trial. The primary objectives of this dose-escalation trial were to determine if OTS11101 given subcutaneously once weekly at 1.0–3.0mg could be confirmed as safe and tolerable treatment, and to collect overall safety data. The secondary objectives included the determination of the MTD, pharmacodynamics, and preliminary information about the antitumor activity and the efficacy on angiogenic peripheral blood biomarkers. The study was reviewed and approved by the Institutional Review Board. OTS11101 was emulsified with Incomplete Freund\'s adjuvant. Dose levels of OTS11101 were 1.0, 2.0, and 3.0mg once weekly in a 28-day cycle. If a patient experienced a drug-related dose-limiting toxicity (DLT), excluding injection site reaction, the treatment with OTS11101 was discontinued. The dose escalation/reduction scheme was based on the occurrence of drug-related DLTs within the first treatment course. If a DLT was not observed in any of the first three patients, the dose was escalated to the next level. If a DLT was observed in one of the first three patients, three additional patients were recruited to that dose level. If a DLT occurred in only one of six patients, dose escalation was permitted. If two or more of six patients experienced a DLT, an independent data monitoring committee determined the dose escalation or reduction decision or stopped the recruitment of additional patients. The safety and tolerability of OTS11101 were assessed according to the Common Toxicity Criteria for Adverse Events version 3.0. The following adverse events were defined as DLTs: grade 4 hematologic and grade 3/4 nonhematologic toxicity except for grade 3 anorexia, nausea, vomiting, diarrhea, constipation, and γ-glutamyl transpeptidase (γ-GTP) increases that could be controlled by supportive treatment. Objective tumor response was evaluated according to the Response Evaluation Criteria in Solid Tumors version 1.1.13 An enzyme-linked immunospot (ELISPOT) assay was performed to measure the specific CTL response against the peptide.14 Peripheral blood mononuclear cells (PBMC) were obtained from patients before the vaccination treatment and at the end of first course. Frozen PBMC cultured with OTS11101 and IL-2 Peptide was added into the culture at days 0 and 7 and cells were harvested after 2weeks. After harvesting, CD4-positive cells were depleted using a Dynal CD4 positive isolation kit (Invitrogen, Carlsbad, Canada) and were used as responder cells in the ELISPOT assay. A human interferon (IFN)-γ ELISpot PLUS kit (MabTech, Nacka Strand, Sweden) was used to measure the CTL responses. HIV-A24 peptide (RYLRDQQLL)-pulsed TISI cells were used as negative control stimulator cells.15 The number of peptide-specific spots was calculated by subtracting the spot number in the control well from the spot number of well with OTS11101-pulsed TISI cells. The CTL response was estimated as positive when the average count of peptide-specific spots was greater than 20 spots/well. The method in this section is described in detail in data S1. The concentrations of serum-soluble VEGFR (sVEGFR) 1 and 2 were measured by ELISA before the vaccination on day 1 and after the vaccination on days 8 and 29 (VERSA max; Molecular devices, Sunnyvale, CA, USA). Furthermore, plasma cytokines related to angiogenesis were measured using an antibody suspension array full system (Bio-Rad Laboratories, Hercules, CA, USA) as previously described16 and included the following: angiopoietin II (ANG-2), follistatin, granulocyte colony-stimulating factor (G-CSF), interleukin-8 (IL-8), leptin, platelet-derived growth factor-BB (PDGF-BB), platelet endothelial cell adhesion molecule-1 (PECAM-1), VEGF, and hepatocyte growth factor (HGF). The PBMC were obtained from whole blood using an ACCUSPIN System (Sigma–Aldrich, St. Louis, MO, USA). The RNA extraction method and the quality check protocol have been described previously.17 The microarray procedure was performed according to the Affymetrix protocols (Affymetrix, Santa Clara, CA, USA) and has been previously described.18 The microarray analysis was performed using the BRB Array Tools software version 4.2.0 Beta 1 (http://linus.nci.nih.gov/BRB-ArrayTools.html). In brief, a log base 2 transformation was applied to the raw microarray data, and global normalization was used to calculate the median over the entire array. Genes were excluded if the percentage of data missing or filtered out exceeded 50%. Genes that passed the filtering criteria were then considered for further analysis. In gene ontology and BioCarta pathway analyses, the statistical significance of global gene expression changes was assessed by mean negative natural logarithm of the P-values of the respective single gene univariate test (LS)/Kolmogorov-Smirnov (KS) permutation tests and the Efron-Tibshirani\'s GSA MaxMean test. Statistical significance levels for gene ontology analysis and pathway analysis were set at P=0.0001 and =0.001, respectively. The gene set comparison tool analyzes pre-defined gene sets for differential expression among pre-defined classes (Pre versus Post). This indicates which gene sets contain more differentially expressed genes than would be expected by chance. \"BioCarta” is a trademark of BioCarta Inc. Student\'s paired t-test was used to compare serum sVEGFR1 and sVEGFR2 levels or plasma cytokine levels at baseline (pre-treatment) and on days 8 and 29, to evaluate the significance of changes induced by OTS11101. A P-value of 0.05 was considered statistically significant. From December 2009 to November 2010, nine HLA-A*24:02-positive patients were treated with OTS11101. The baseline demographics of these patients are shown in Table1. All patients had previously received one or more chemotherapy regimens for advanced disease. All patients completed the first cycle of four injections of OTS11101 and six patients were subjected to further cycles of vaccination. The median (range) number of vaccinations in the whole cohort was nine (4–44). All nine patients received at least one dose of study treatment and were evaluated for safety (Table2). One patient with cholangiocellular cancer given a dose of 2.0mg experienced a grade 3 γGTP increase. His hepatic metastases had progressed and invaded the common bile duct; therefore, the investigators and independent data monitoring committee judged the γGTP increase as an adverse event related to the primary disease and agreed to escalate to the third dose level of 3.0mg. No other patients showed any toxicities of grade 3 or greater. Three patients (33.3%) (two in the 1.0-mg cohort and one in the 2.0-mg cohort) developed injection site reactions (grade 1). In the 1.0-mg cohort, one patient reported grade 2 proteinuria. Grade 2 hypertension was identified in a patient receiving the 3.0-mg dose. No DLTs were observed in this trial. Presented is the highest ever reached CTCAE grade. One patient may have experienced 1 event. CTCAE, common terminology criteria for adverse events; LDH, lactase dehydrogenase increased; γ-GTP, γ-glutamyl transferase; TAT, thrombin-antithrombin complex. All patients were evaluated for tumor response. Although no complete or partial responses were observed, five patients (55.5%) had stable disease (SD) for at least one treatment courses (28days). The median duration of progression free survival (PFS) for all patients was 50days (range: 31–201 days). An IFN-γ ELISPOT assay was conducted using PBMC periodically obtained from patients to assess the cellular immune responses to OTS11101. Positive CTL responses specific to the vaccinated peptide were determined as described in the 2 section. Positive CTL responses were seen in one of the three patients in both the 2.0 and 3.0-mg dose cohorts (Table3, Fig. S1). The positive CTL response in the 2.0-mg cohort was seen in a 68-year-old female patient with follicular thyroid cancer who had multiple metastases in the lungs. She achieved stable disease that persisted for 3 months after initiating the vaccination. A 62-year-old female patient with uterine cervix cancer receiving 3.0mg who had multiple metastases in the lungs also showed a positive CTL response. As the tumor continued to grow, despite chemotherapy, she was enrolled in this study. After initiating the vaccination, the tumor size evaluated by computed tomography remained stable for 6months. GIST, gastrointestinal stromal tumor; NA, not analyzed; PD, progression disease; PFS, progression free survival; SD, stable disease. Serum levels of sVEGFR1, sVEGFR2, and other plasma cytokines related to angiogenesis during OTS11101 peptide vaccination We investigated serum sVEGFR1, sVEGFR2, and other plasma cytokines related to angiogenesis as biomarker for OTS11101 vaccination (Fig.1). The serum concentrations of sVEGFR1 on day 29 increased significantly over the first 4weeks of treatment (P 0.001, t-test). The serum concentrations of sVEGFR2 were also upregulated on days 8 and 29 (P 0.016 and 0.044, respectively). Greater increases in sVEGFR1 levels on day 8 were seen at the higher dose levels of OTS11101. Figure 1Open in figure viewerPowerPoint Changes in angiogenesis-related biomarker concentrations in the blood after OTS11101 vaccinations: Evaluated at pretreatment, and after one and four vaccinations. The serum concentrations of serum-soluble vascular endothelial growth factor receptor 1 (sVEGFR1) increased significantly over the first 4weeks of treatment (A). The serum concentrations of sVEGFR2 were also upregulated on days 8 and 29 (B). The plasma concentrations of VEGF (C), interleukin (IL)-8 (D), and platelet endothelial cell adhesion molecule-1 (PECAM-1) (E) were significantly elevated on day 29 compared with the baseline. The experiment was performed in triplicate. Additionally, according to the analysis of angiogenesis-related cytokines, the level of IL-8, PECAM-1, and VEGF were significantly elevated on day 29 compared with the baseline (Fig.1, Fig. S2). To determine whether the OTS11101 vaccination induced any systemic immunological effects, we examined the gene expression profiles of PBMC of all nine patients on days 1 and 8, using microarray analysis. A gene set analysis was performed by comparing the overall significance of gene groups defined by GO categories. Out of the 4121 GO classes, 281 were found to be significantly affected by OTS11101 vaccination, as shown by at least one of the three tests used to assess the significance of the differences (Table S1). Six out of the 280 cellular component categories, 24 of 655 molecular function (MF) categories and 251 of 3186 biological process (BP) categories were significant. Interestingly, the gene ontology categories of \"T cell activation” and \"immune response” were selected. These results suggested that the genes related to T cell activation or immune response might be associated with vaccination response. Furthermore, pathway analysis of gene expression microarray data revealed that 19 pathways were significantly selected from 288 BioCarta pathways at the nominal 0.01 level of the LS permutation test or KS permutation test than expected by chance (Table4). The most represented classes of differently expressed genes included genes involved in T cell function-related pathways, strongly supporting the notion that OTS11101 vaccination actually gives rise to T cell activation in clinical settings. Data considered significant (P 0.001) are shown in bold. CCR5, C-C chemokine receptor type 5; CREM, cyclic AMP response element modulator; CTL, cytotoxic T cell lymphocyte; D4-GDI, Rho GDP dissociation inhibitor beta; HIV, human immunodeficiency virus; KS, Kolmogorov-Smirnov; LS, mean negative natural logarithm of the P-values of the respective single gene univariate test; NK, natural killer; TSP-1, thrombospondin-1. To our knowledge, the clinical trial described here is the first human trial to consist of a vaccine strategy that has used the HLA-A*24:02-restricted epitope-peptide derived from VEGFR1 (VEGFR1-1084: OTS11101). The current phase I study showed that OTS11101 can be safely given to patients with advanced solid tumors, and no DLT was observed in any cohort. Analysis of the ELISPOT assay results indicated that peptide-specific CTL could be induced by the OTS11101 vaccination. Furthermore, biomarker analysis provided evidence that OTS10111 treatment actually provoked an anti-angiogenic effect and T cell activation in a clinical setting. Targeting tumor angiogenesis with vaccines has potential advantages over targeting tumor cells. First, tumor endothelial cells are more accessible to the immune system than are tumor cells at a distance from the vessels.19 Cancer endothelial cells are readily accessed by lymphocytes in the bloodstream, and CTLs can directly damage endothelial cells without the penetration of any other tissue type. In addition, the lysis of even a small number of endothelial cells within the tumor vasculature may result in the destruction of vessel integrity, thus leading to the inhibition of numerous tumor cells.20 Therefore, endothelial cells could be a good target for cancer immunotherapy. Second, the loss or downregulation of HLA molecules on tumor cells is considered to be one of the major reasons for limited clinical efficacy.21-23 Since such HLA loss has not been reported for endothelial cells of newly formed vessels in tumors, the development of vaccines against vascular endothelial cells in tumor tissues may overcome the immune-escape of tumor cells. In the present trial, we observed no severe adverse effects related to the treatment; as anticipated, patients experienced local injection site reactions, and a few experienced low-grade constitutional symptoms such as fever and fatigue. With respect to the specific toxicity of anti-angiogenic treatment, such as hypertension, proteinuria, and bleeding, which is often observed with anti-VEGF antibody and VEGF tyrosine kinase receptor inhibitors,24, 25 low-grade toxicity occurred in two patients (grade 2 proteinuria and hypertension in each, respectively). VEGFR1 has multiple functions not only in angiogenesis but also in hematopoiesis. Previous studies have reported that VEGFR1 is expressed on hematopoietic stem cells and that it plays functional roles in the recruitment of hematopoietic stem cells and reconstitution of hematopoiesis.26 However, our results showed that bone marrow hematopoiesis was not affected by immunization with OTS11101 in a clinical setting. Thus, this vaccination is considered to be very safe and well tolerated compared with existing anti-angiogenic treatments. In addition, the current trial demonstrated that clinical SD was achieved in five different types of cancer in which the VEGF pathway plays a potentially major role for angiogenesis or tumor progression.27-32 We were unable to reach any firm conclusion from the small size of this phase I trial; therefore, further studies are required to investigate the efficacy of OTS11101 vaccination. Proof of concept (POC) based on immunologic monitoring is crucial for the rational design of cancer vaccination studies. Peptide vaccine trials have focused predominately on stimulating CTLs, through the administration of 8–10 mers binding on HLA class I alleles and such vaccine-induced CTLs can directly kill tumor cells.33 The specific CTL responses against the OTS11101 vaccination were observed at 2-mg and 3-mg dose levels, clearly demonstrating that CTLs against VEGFR1 could be induced by the vaccination. Additionally, it is important to address the toxicity to endogenous antigen-expressing cells. In the current study, we did not monitor the CTL response against the endogenous target. However, a previous study demonstrated that the CTLs induced with the VEGFR1 peptide induced strong cytotoxicity against target cells endogenously expressing VEGFR1.12 Several parameters measured in the blood circulation of patients with cancer might hold pharmacodynamic biomarker value. Naturally, VEGF, which is the most extensively explored biomarker, has been examined in numerous clinical trials. The present study demonstrated that serum VEGF levels were elevated after OTS11101 vaccination. The increases in these key mediators of angiogenesis are similar to those seen in the study of VEGF TKIs and anti-VEGF antibody in cancer patients or in preclinical models.34-38 We also observed a time-dependent change in serum sVEGFR1 and sVEGFR2 levels. Several clinical trials have detected changes in the serum levels of sVEGFR1 and sVEGFR2.39 In contrast to the VEGFR TKIs, all of which significantly decrease sVEGFR2 levels,34, 36, 40-45 OTS11101 induced a mild but significant increase in serum sVEGFR1 and sVEGFR2. OTS11101 acts as an angiogenesis inhibitor by inducing CTLs that target vascular endothelial cells expressing VEGFR1, whereas VEGFR TKIs and anti-VEGF antibody directly inhibit the VEGF/VEGFR pathway. We speculate that this difference in pharmacological function between the vaccination and existing VEGF/VEGFR targeted agents creates such discrepancies in the kinetics of sVEGFR1 and sVEGFR2. Although the biological significance of this is not entirely understood, it suggests that OTS11101 potentially possesses a distinct antitumor mechanism of action compared with VEGFR TKIs. Furthermore, OTS11101 modulated the plasma concentration of inflammatory cytokines such as IL-8 and PECAM-1, the latter of which is a member of the immunoglobulin superfamily of cell surface receptors. A limitation of biomarker analysis in the present study is the lack of examination of the vasculature in tumor samples. Investigation of changes to the vasculature in tumor tissues after vaccination is an attractive approach to POC, and several studies have reported a reduction in microvessel density in tumor tissues of patients receiving bevacizumab.46-48 In future clinical trials evaluating the efficacy of OTS11101 vaccination, an investigation of the changes to the vasculature in tumor samples is needed. While ex vivo or in vitro studies have provided a wealth of information on the specific effect of immuno-peptide therapy, they cannot substitute for an ultimate understanding of what goes on in vivo. There is no valid and widely accepted in vivo analysis to date to achieve POC during the clinical development of cancer vaccines. Microarray technology has enabled significant genes to be identified almost throughout the genome using a hypothesis-free approach. The recent introductions of this technology in the field of cancer research are numerous, providing insights into a more accurate classification of cancer, better defined prognosis, and novel approaches for therapy. Microarray analyses have been shown to be powerful tools in identifying and characterizing gene regulation in the ontogeny, differentiation, and activation of immune cells. In the present study, we have used the microarray procedure using PBMC obtained from patients to monitor the biological activity of OTS11101. To effectively interpret the enormous amount of microarray data, we examined biologically relevant gene pathways rather than individual genes. Although it is difficult to determine whether the observed pathways elicited by OTS11101 vaccination led to the activation of CTLs or the suppression of regulatory T-cells, we detected several pathways related to CTL response. For example, in the CTL-mediated immune response against target cell pathways, a set of genes identified as significantly upregulated following OTS11101 vaccination included granzyme B, perforin, CD247, FAS ligand, ICAM1, and integrin. It is known that pathways containing cytotoxic proteins, such as perforin and granzyme B can mediate cancer cell death via CTLs. FAS-ligand induced apoptosis also plays a crucial role in the CTL response. Thus, the results of our analysis indicate that several pathways related to cytotoxic function of CTL were significantly affected by a single administration of OTS11101, supporting the notion that this peptide induces an immune response. Our current study introduced a novel direction in which microarray analysis could be useful for achieving POC during early clinical trials of cancer vaccine. In conclusion, OTS11101 shows an acceptable safety profile for patients with advanced solid tumors. The preliminary evaluations of biological activity of OTS11101 using microarray and biomarker analysis demonstrate that this vaccine is biologically active. Further trials to evaluate the efficacy of this promising vaccination are warranted, and phase II clinical trial evaluating the anti-tumor activity of OTS11101 in combination with gemcitabine in patients with locally advanced or metastatic pancreatic cancer are ongoing. We thank Dr Richard Simon and BRB-ArrayTools Development Team for providing us with the BRB ArrayTools software. Takuya Tsunoda and Koji Yoshida led the study and were employed by Onco Therapy Science Inc. Taroh Satoh has been reimbursed by the following companies for attending several conferences: Chugai Pharmaceutical, Merck-Serono, Bristol-Myers Squibb. Taroh Satoh received honoraria for writing promotional material for Chugai Pharmaceutical, Merck-Serono and Bristol-Myers Squibb. Takayasu Kurata received honoraria for a lecture fee for Eli Lilly and AstraZeneka. Other authors have no conflict of interests. This study was funded by Otsuka Pharmaceutical Company. cas12034-sup-0002-FigureS2.tifimage/tif, 116.6 KB Fig. S2. Changes in angiogenesis-related cytokine concentrations. cas12034-sup-0004-TableS1.docxWord document, 59.3 KB Table S1. Assessment of overall significance of expression changes in gene groups defined by Gene Ontology (GO) categories. cas12034-sup-0003-DataS1.docxWord document, 15.9 KB Data S1. Methods of measurement of CTL responses in detail. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. 1Taylor M, Rossler J, Geoerger B etal. 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