Phase II clinical trial of peptide cocktail therapy for patients with advanced pancreatic cancer: VENUS-PC study.

We previously conducted a phase I clinical trial combining the HLA-A*2402-restricted KIF20A-derived peptide vaccine with gemcitabine for advanced pancreatic cancer (PC) and confirmed its safety and immunogenicity in cancer patients. In this study, we conducted a multicenter, single-armed, phase II trial using two antiangiogenic cancer vaccines targeting VEGFR1 and VEGFR2 in addition to the KIF20A peptide. We attempted to evaluate the clinical benefit of the cancer vaccination in combination with gemcitabine. Chemotherapy naïve PC patients were enrolled to evaluate primarily the 1-year survival rate, and secondarily overall survival (OS), progression free survival (PFS), response rate (RR), disease control rate (DCR) and the peptide-specific immune responses. All enrolled patients received therapy without the HLA-A information, and the HLA genotypes were used for classification of the patients. Between June 2012 and May 2013, a total of 68 patients were enrolled. No severe systemic adverse effects of Grade 3 or higher related to these three peptides were observed. The 1-year survival rates between the HLA-A*2402-matched and -unmatched groups were not significantly different. In the HLA-A*2402 matched group, patients showing peptide-specific CTL induction for KIF20A or VEGFR1 showed a better prognosis compared to those without such induction (P = 0.023, P = 0.009, respectively). In the HLA-A*2402-matched group, the patients who showed a strong injection site reaction had a better survival rate (P = 0.017) compared to those with a weak or no injection site reaction. This phase II study demonstrated that this therapeutic peptide cocktail might be effective in patients who demonstrate peptide-specific immune reactions although predictive biomarkers are needed for patient selection in its further clinical application.

[Adoptive Cell Therapy with Immune Checkpoint Blockade].

Cancer immunotherapy are taking a leading role of cancer therapy due to the development of the immune checkpoint blockade. To date, however, only about 20% of patients have clinical responses and the cancer-specific T cells in cancer site are required to obtain beneficial effects. There has been an innovative development in the field of adoptive cell therapy, especially receptor gene-modified T cells in recent years. The effector cells mostly express PD-1, therefore the cytotoxic reactivity of the effector cells are inhibited by PD-L1. The combination of the adoptive cell therapy and the immune checkpoint blockade is expected to enhance efficacy. On the other hand, the immune-related adverse events may also be enhanced, therefore, it is needed to develop the combination therapy carefully, improving the cancer antigen-specificity or dealing with the cytokine release syndrome.

Phase I clinical trial of multiple-peptide vaccination for patients with advanced biliary tract cancer.

BACKGROUND: The prognosis of patients with advanced biliary tract cancer (BTC) is extremely poor and only a few standard treatments are available for this condition. We performed a phase I trial to investigate the safety, immune response and anti-tumor effect of vaccination with three peptides derived from cancer-testis antigens. METHODS: This study was conducted as a phase I trial. Nine patients with advanced BTC who had unresectable tumors and were refractory to standard chemotherapy were enrolled. Three HLA-A*2402 restricted epitope peptides-cell division cycle associated 1 (CDCA1), cadherin 3 (CDH3) and kinesin family member 20A (KIF20A)-were administered subcutaneously, and the adverse events and immune response were assessed. The clinical effects observed were the tumor response, progression-free survival (PFS) and overall survival (OS). RESULTS: The three-peptide vaccination was well-tolerated up to a dose of 3 mg per peptide (9 mg total). No grade 3 or 4 adverse events were observed after vaccination. Peptide-specific T cell immune responses were observed in all patients and stable disease was observed in 5 of 9 patients. The median PFS and OS were 3.4 and 9.7 months. The Grade 2 injection site reaction and continuous vaccination after PD judgment appeared to be prognostic of OS. CONCLUSIONS: Multiple-peptide vaccination was well tolerated and induced peptide-specific T-cell responses. TRIAL REGISTRATION: This study was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR000003229).

[Establishment of induced pluripotent stem cells from adipose tissue-derived stem cells for dendritic cell-based cancer vaccines].

Recently, studies on regenerative stem cell therapy are being encouraged, and efforts to generate dendritic cells, which play important roles in cancer immunotherapy, from stem cells are being made in the field of tumor immunology. Therapeutic acquisition of stem cells has important clinical applications. Studies on induced pluripotent stem(iPS)cells generated from somatic cells with pluripotent genes have advanced in recent years. Stem cells are reported to be found in adipose tissue (adipose-derived stem cells, ADSC). Our goal is to develop a new cancer vaccine by using dendritic cells generated from ADSC. In a preliminary study, we examined whether iPS cells can be generated from ADSC to serve as a source of dendritic cells.We introduced a plasmid with pluripotent genes(OCT3/4, KLF4, SOX2, L-MYC, LIN28, p53-shRNA)into an ADSC strain derived from adipose tissue by electroporation and subsequently cultured the cells for further examination. A colony sugges- tive of iPS cells from ADSC was observed. OCT3/4, KLF4, SOX2, L-MYC, and LIN28 mRNAs were expressed in the cultured cells, as confirmed by reverse transcriptase-polymerase chain reaction(RT-PCR). On the basis of these results, we confirmed that iPS cells were generated from ADSC. The method of inducing dendritic cells from iPS cells has already been reported, and the results of this study suggest that ADSC is a potential source of dendritic cells.

Analysis of Off-target Effects and Risk Assessment Leading from Preclinical to Clinical Trials of Gene-edited Therapeutic Products.

OBJECTIVE: Concerns about off-target effects (OTEs) of genomic DNA cleavages by gene-editing enzymes have been raised, especially for OTEs that go undetected due to technical limitations. Since no explicit guidelines have been in place for risk assessment of OTEs, the regulatory authorities' concept of an acceptable evaluation scheme for OTEs in the investigational drug application (IND) has not been clear. Here, we clarify the regulatory expectations by examining reports on OTE evaluations of leading gene-editing products that have achieved IND clearance. METHODS: We collected and analyzed the gene-editing products that have entered clinical trials by searching on ClinicalTrials.gov and EU-Clinical-Trial-Registries, and related reports for OTE evaluations from Google Scholar, PubMed, and the developers' websites. RESULTS: We found a common two-step verification method used for different products at the preclinical stage. First, numerous potential off-target loci (POLs) are listed with state-of-the-art high-sensitivity detection methods and theoretical screens; Second, these OTEs are checked by amplicon sequencing of the POLs after treatment by enzymes in in vitro models close to clinical use conditions. Only the OTEs that can be detected and verified are addressed in the risk assessment in the translational phase from preclinical to clinical study. DISCUSSION: Here, we describe a clear scheme for risk assessment of OTEs at the key translational phase, based on the common features in protocols for gene-editing products that were cleared for use in clinical trials. This report will provide a guide for those newly attempting to conduct clinical development in this field.

[Induction of human dendritic cells from peripheral blood mononuclear cells by using a HydroCellTM].

Cancer immunotherapy using dendritic cells (DCs) is widely known to produce an anticancer effects. However, there is no approved DC vaccine in Japan, possibly because the culture and harvesting of DC is complicated. To further enable DC vaccine therapy, an easier, safer, and inexpensive culture system is needed, and we therefore aimed to build an automated DC culture system. In a previous study using a HydroCellTM, we successfully cultured and harvested DCs from peripheral blood monocytes without using enzymes, and showed that HydroCellTM is a useful tool for automated DC culture. In this study, we performed a similar process of DC culture from peripheral blood mononuclear cells and demonstrated the safe and easy culturing and harvesting of DCs. Furthermore, CD83-positive mature DCs were increased compared to the usual plate method. These results demonstrate the utility of HydroCellTM as an automated DC culture system.

[In vitro culture of human dendritic cells by using a HydroCell™].

Cancer Immunotherapy using dendritic cells would be a feasible and useful tool for cancer treatment. However, no immunotherapy has been approved in Japan because of a lack of any randomized clinical studies. We are now trying to develop an automatic dendritic cell culture system in order to perform a large-scale randomized clinical trial. In this study, we investigated the utility of a HydroCell™ for in vitro culture of human dendritic cells induced from peripheral blood monocytes. The dendritic cells grew one and a half times when they were cultured in a HydroCell™. All the cells were floating and harvested easily without any enzymes. The cells expressed the CD80 and CD83 molecules on their surface and still had strong phagocytosis. This results demonstrated that a HydroCell™ was a useful tool for in vitro culture of dendritic cells.

[Dendritic cell-based cancer immunotherapy].

Dendritic cell is the most effective antigen presenting cells in human. We performed a couple of dendritic cell-based cancer immunotherapy in Tokyo Women's Medical University. Autologous tumor lysates or synthetic peptides are pulsed with dendritic cells to make the dendritic cell vaccine. Tumor-pulsed dendritic cell vaccine (TP-DC) or peptide pulsed dendritic cell vaccine (PP-DC) are safe and useful in the patients with advanced cancer. Antigen unpulsed immature dendritic cell could work at a cancer vaccine when they inject into tumors in vivo. Dendritic cell-activated T cells (DCAT) in vitro are also useful tools for cancer treatment. In order to obtain a good evidence of dendritic cell-based immunotherapy, we need to perform the randomized trial near the future.

[Dendritic cell vaccine for intrahepatic cholangio cellular carcinoma--a study of relationship between immuno-reaction and clinical outcome].

The clinical outcome of intra-hepatic cholangiocelluler carcinoma (ICC) is very poor. To prevent a tumor relapse after curative operation, we employed the combination therapy of adaptive transfer of anti-CD3 activated T cells and dendritic cell vaccine therapy (DC-CAT therapy) since 2000. During DC-CAT therapy, about a half of patient revealed skin reaction positive. Moreover, the patient who had a skin reaction showed a significantly good clinical outcome. In this study, we investigated the difference between skin reaction positive and negative checking with the surface markers of T cells. The patient who had skin reaction positive increased the number of lymphocyte. Especially, CD8 positive T cells were increased about 2 folds in number before the DC-CAT therapy, and decreased Foxp3 positive regulatory T cells. Our data suggest that increasing cytotoxic T cells and decreasing regulatory T cells made the difference of the clinical outcome such as a disease free survival rate and overall survival rate after curative operation for ICC patient.

[The case of tumor escape mechanism by changing their tumor-associated antigens].

A 53-year-old woman presented with a diagnosis of advanced gallbladder cancer at our hospital. She was evaluated with CT scan and given a diagnosis of Stage IVb due to the multiple lymph nodes metastases and significant invasion to the artery. However, we underwent simple cholecystectomy followed by immunotherapy that was the hope of herself and her family. The serum level of DUPAN-2 was gradually elevated to 6,800 U/mL, and the metastases to the liver were detected. After we started the dendritic cell vaccine pulsed with autologous tumor-lysate with S-1, DUPAN-2 decreased to 980 U/ mL. The CT scan showed complete response (CR) in the liver metastases and partial response (PR) in the lymph node metastases. However, the serum level of CEA elevated since the MUC-1 peptide was used instead of autologous tumor- lysate, even DUPAN-2 did not. The liver metastases were in control, but the lymph nodes metastases had progressed. She died of the progressed lesion later in approximately one year from the operation. This case demonstrated a possibility of the tumor escape mechanism by changing their tumor-associated antigens.

[The analysis of CCR4+/CXCR3+ cell ratio in gastrointestinal cancer patients receiving chemotherapy or immunotherapy].

We investigated the expression of CCR4 and CXCR3 with the peripheral blood lymphocytes in 31 gastrointestinal cancer patients who received either chemotherapy or immunotherapy. The expressions of CXCR3 were 6.8% in the patients received chemotherapy and 24.2% in the patients received immunotherapy. The expressions of CCR4 were 25.6% in the patients received chemotherapy and 15.4% in the patients received immunotherapy. The ratio of CCR4+/CXCR3+ cells was 4.45 in chemotherapy and 0.72 in immunotherapy. These results suggested that it was the Th2 dominant condition in the patients received chemotherapy and the Th1 dominant condition in the patients received immunotherapy.

A Comparative Safety Profile Assessment of Oncolytic Virus Therapy Based on Clinical Trials.

Oncolytic virus therapy (OVT) represents a new class of therapeutic agents in cancer treatment. The molecular and cellular mechanisms of action of OVTs have been evaluated in nonclinical/clinical phase trials. Various genetically modified viruses have been developed as oncolytic agents, and the first approval of an OVT for clinical use was issued by the US Food and Drug Administration in 2015. In this context, more and more clinical development of OVTs is anticipated in the future. This article provides a risk assessment of OVT based on the safety data obtained from all clinical trials to date using a publicly available database. The most common adverse events (AEs) observed in clinical trials have been infection-related symptoms such as fatigue, chills, fever, and nausea; few serious AEs have been observed, regardless of the kind of virus or transfected genes. In vivo systemic infusion of OVTs demonstrated a high percentage of AEs, but most AEs were manageable using common drugs. This paper describes OVTs' specific safety/toxicity profiles and encourages the performance of further clinical trials of OVTs to address the most serious challenges anticipated in the development of OVTs as a new class of drugs for the treatment of cancer.

In Vivo Antitumor Effects of MK615 Led by PD-L1 Downregulation.

BACKGROUND/AIM: MK615 extracted from Prunus mume was reported to have anti-inflammatory effects. In this article, we examined the in vivo antitumor effect of MK615 (an extract from Japanese apricot) using mouse tumor xenografts and focusing on the downregulation of PD-L1 (programmed death-ligand 1), a ligand of programmed cell death-1, a surface protein of activated T cells. MATERIALS AND METHODS: B16/BL6 melanoma cells were injected into C57BL/6 or BALB/c-nu/nu mice to establish lung metastasis. BALB/c-nu/nu mice (nude mice) were used as a T cell-deficient model. The mice were given MK615 or saline orally every other day for approximately 8 weeks, and their survival was observed. NF-κB (nuclear factor-κB) and PD-L1 expressions of metastatic lung tissues were also examined. RESULTS: The survival rate was improved only in the MK615-treated C57BL/6 mice ( P < .05), not in the saline-given control mice or BALB/c-nu/nu mice. The downregulations of NF-κB and PD-L1 were observed in both MK615-treated C57BL/6 and BALB/c-nu/nu mice. These results suggest that the antitumor effects of MK615 are associated with T cell-mediated immunity activated by MK-615-induced PD-L1 downregulation in tumor cells. CONCLUSION: MK615 is beneficial for a prolonged host survival time in the B16/BL6 melanoma xenograft model associated with T cell-mediated antitumor immunity.

RT-qPCR analysis of the tumor antigens TOMM34 and RNF43 in samples extracted from paraffin-embedded specimens of colorectal cancer.

Comprehensive genetic analysis of colorectal malignant tumors by microarrays has identified translocase of the outer mitochondrial membrane 34 (TOMM34) and ring finger protein 43 (RNF43) as highly expressed oncogenes in malignant colorectal tumors. Vaccine therapy using cancer peptides synthesized using the amino acid sequences of tumor antigens is currently undergoing clinical trials. Since it is important to perform vaccine therapy based on the oncogene expression levels in individual tumors, analysis of tumor antigen expression is necessary for this therapy. However, the quality of the messenger RNA extracted from formalin-fixed and paraffin-embedded specimens is generally considered insufficient for gene quantification. The present study examined whether it could be possible to quantify the expression of TOMM34 and RNF43 in colorectal cancer and liver metastasis samples prepared from paraffin blocks. The formalin-fixed and paraffin-embedded specimens were sliced for slides and the colorectal cancer and normal mucosal tissues were obtained from the slides. Total RNA was extracted from the tissue samples, and quantitative polymerase chain reaction (qPCR) was performed using the Universal ProbeLibrary as a PCR probe. Quantification of TOMM34 and RNF43 gene expression in several-year-old paraffin-embedded colorectal cancer specimens was possible by qPCR using the Universal ProbeLibrary. qPCR revealed that TOMM34 expression was elevated in 78.9% (15 of 19 cases) of the primary tumors and in 73.7% (14 of 19 cases) of the liver metastasis samples. RNF43 expression was elevated in 63.2% (12 of 19 cases) of the primary tumors and in 73.7% (14 of 19 cases) of the liver metastasis samples.

Predictive biomarkers for the efficacy of peptide vaccine treatment: based on the results of a phase II study on advanced pancreatic cancer.

BACKGROUND: The purpose of the present study was to explore novel biomarkers that can predict the clinical outcome of patients before treatment or during vaccination. These would be useful for the selection of appropriate patients who would be expected to exhibit better treatment outcomes from vaccination, and for facilitating the development of cancer vaccine treatments. METHODS: From a single-arm, non-randomized, human leukocyte antigen (HLA)-A-status-blind phase II trial of a vaccine treatment using three HLA-A*2402-restricted peptides for advanced pancreatic cancer (PC), we obtained peripheral blood samples from 36 patients of an HLA-A*2402-matched group and 27 patients of an HLA-A*2402-unmatched group. RESULTS: Multivariate analysis (HR = 2.546; 95% CI = 1.138 to 5.765; p = 0.0231) and log-rank test (p = 0.0036) showed that a high expression level of programmed death-1 (PD-1) on CD4+ T cells was a negative predictive biomarker of overall survival in the HLA-A*2402-matched group . Moreover, a high expression level of PD-1 on CD4+ T cells was a negative predictor for the induction of cytotoxic T lymphocytes (p = 0.0007). After treatment, we found that the upregulation of PD-1 and T cell immunoglobulin mucin-3 (Tim-3) expression on CD4+ and CD8+ T cells was significantly associated with a poor clinical outcome in the HLA-A*2402-matched group (p = 0.0330, 0.0282, 0.0046, and 0.0068, respectively). In contrast, there was no significant difference for these factors in the HLA-A*2402-unmatched group. CONCLUSIONS: Our results indicate that the upregulation of PD-1 and Tim-3 expression on CD4+ and CD8+ T cells may restrict T cell responses in advanced PC patients; therefore, combination immunotherapy with blockade of PD-1 and Tim-3 to restore T cell responses may be a potential therapeutic approach for advanced PC patients. TRIAL REGISTRATION: Clinical-Trail-Registration: UMIN000008082 .

OK432-activated natural killer cells enhanced trastuzumab (Herceptin)-mediated antibody-dependent cellular cytotoxicity in patients with advanced cancer.

BACKGROUND: Adoptive immunotherapy using natural killer (NK) cells from cancer patients yielded only modest success. Whether Herceptin and OK432-activated NK cells (NK cells obtained by stimulating peripheral blood mononuclear cells with OK432 and interleukin-2) improve the outcome of adoptive immunotherapy against HER-2/neu positive tumor cells via antibody-dependent cellular cytotoxicity, was examined in vitro. MATERIALS AND METHODS: A 51Cr release assay was performed to assess cytotoxicity. OK432-activated NK cells and lymphokine-activated killer (LAK) cells from healthy donors and cancer patients were used as effectors. Three cell lines expressing different amounts of HER-2/neu served as targets. RESULTS: In the case of effectors from patients, OK432-activated NK cells showed higher cytotoxicity than that of LAK cells, and the cytotoxicity of OK432-activated NK cells against the SK-BR-3 cell line (over-expressing HER-2/neu) was increased by Herceptin. CONCLUSION: Combination of Herceptin and OK432-activeted NK cells may improve the efficacy of the treatment for HER-2/neu-positive malignancy.

Applicability of Selective Data Collection to Cancer Clinical Studies for Supplemental Marketing Approvals: Frequency of Adverse Reactions Observed During Supplemental Approval Compared With First Approval.

BACKGROUND: In 2012, the US Food and Drug Administration (FDA) issued the draft guidance "Determining the Extent of Safety Data Collection Needed in Late Stage Premarket and Postapproval Clinical Investigations." The selective data collection approach proposed in this guidance leads to reductions in costs and work time and may improve the quality of the database for clinical trials. The current study evaluated the applicability of selective data collection for oncology drugs. METHODS: The labeling information of oncology drugs obtained supplemental approvals from the FDA between 2005 and 2014 were used. The frequency of adverse reactions observed in clinical trials between the first approval and supplemental approvals of a specific drug were compared. Paired studies were categorized into the following 4 groups: A, same tumor type and same usage; B, same tumor type and different usage; C, different tumor type and same usage; D, different tumor type and different usage. RESULTS: A total of 46 study pairs for additional drug indications were investigated. In group A, 6 of the 7 pairs showed a high correlation coefficient ( r = 0.988, 0.953, 0.947, 0.935, 0.853, and 0.846). CONCLUSIONS: Selective data collection should be adopted in cases in which the additional indication of a drug is for the same tumor type and usage as the first or previous indication.

Compassionate use of drugs and medical devices in the United States, the European Union and Japan.

Compassionate use, also called expanded access, provides an important pathway for patients with life-threatening conditions to gain access to unapproved investigational drugs, biologics and medical devices. Although the United States (US) and the countries of the Europe Union (EU) have mechanisms that are associated with the use of unapproved products, as of May 2015 there was no such mechanism in Japan. Instead, unapproved products are used under a physician's discretion in conjunction with the Japan Medical Practitioners' Act or Advanced Medical Care B. However, there are some issues and questions to consider under the current circumstances in Japan as follows: (A) it is difficult for the local regulator to monitor the use of unapproved products; (B) there is no information collected on the safety of these products to protect patients; (C) it is difficult to assure the quality of the products; (D) it is difficult for patients to obtain detailed information about unapproved products and their availability; and (E) it is not clear who should cover the cost of the unapproved products. In this paper, we assess the current compassionate use, or expanded access-related mechanisms, of the US, the EU and Japan in regard to drugs, medical devices and biologics, including human cells and tissue products, and discuss the benefits and issues of these mechanisms. The purpose of these mechanisms is principally to save patients with life-threatening condition. However, the information obtained after the compassionate use is potentially useful to facilitate marketing authorization. In fact, the data from compassionate use cases are employed in some approval review reports to indicate the product safety.