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.

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.

[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.

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 .

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.

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.

Comparison of Current Regulatory Status for Gene-Based Vaccines in the U.S., Europe and Japan.

Gene-based vaccines as typified by plasmid DNA vaccines and recombinant viral-vectored vaccines are expected as promising solutions against infectious diseases for which no effective prophylactic vaccines exist such as HIV, dengue virus, Ebola virus and malaria, and for which more improved vaccines are needed such as tuberculosis and influenza virus. Although many preclinical and clinical trials have been conducted to date, no DNA vaccines or recombinant viral-vectored vaccines expressing heterologous antigens for human use have yet been licensed in the U.S., Europe or Japan. In this research, we describe the current regulatory context for gene-based prophylactic vaccines against infectious disease in the U.S., Europe, and Japan. We identify the important considerations, in particular, on the preclinical assessments that would allow these vaccines to proceed to clinical trials, and the differences on the regulatory pathway for the marketing authorization in each region.

Approaches to improve development methods for therapeutic cancer vaccines.

Therapeutic cancer vaccines are an immunotherapy that amplify or induce an active immune response against tumors. Notably, limitations in the methodology for existing anti-cancer drugs may subsist while applying them to cancer vaccine therapy. A retrospective analysis was performed using information obtained from ClinicalTrials.gov, PubMed, and published articles. Our research evaluated the optimal methodologies for therapeutic cancer vaccines based on (1) patient populations, (2) immune monitoring, (3) tumor response evaluation, and (4) supplementary therapies. Failure to optimize these methodologies at an early phase may impact development at later stages; thus, we have proposed some points to be considered during the early phase. Moreover, we compared our proposal with the guidance for industry issued by the US Food and Drug Administration in October 2011 entitled "Clinical Considerations for Therapeutic Cancer Vaccines". Consequently, while our research was aligned with the guidance, we hope it provides further insights in order to predict the risks and benefits and facilitate decisions for a new technology. We identified the following points for consideration: (1) include in the selection criteria the immunological stage with a prognostic value, which is as important as the tumor stage; (2) select immunological assays such as phenotype analysis of lymphocytes, based on their features and standardize assay methods; (3) utilize optimal response criteria for immunotherapy in therapeutic cancer vaccine trials; and (4) consider supplementary therapies, including immune checkpoint inhibitors, for future therapeutic cancer vaccines.

New concepts of biomarkers and clinical outcomes for therapeutic cancer vaccines in clinical trials.

AIM: This study aimed to derive meaningful parameters for immune monitoring during cancer vaccine development by analysis of the literature. METHODS: This retrospective study was based on analysis of clinical trials registered at ClinicalTrials.gov and published data available on PubMed. RESULTS: The most common sample evaluated in immune monitoring was peripheral blood. All trials employed ELISA for detecting a humoral immune response; however, cellular immune assays were not used across trials. Most cellular immune assays failed to correlate with clinical outcome, although results of other methods did. CONCLUSION: Standardization of the cellular immune assays across trials is important for predicting the effects of therapeutic cancer vaccines when considering the reliability and characteristics of the methods. Currently, assays mostly target detection of T-cell function, such as proliferation and cytokine release; however, T-cell phenotype analysis in peripheral blood and/or tumor sites may also be considered in the future.

Fever-range whole-body heat treatment stimulates antigen-specific T-cell responses in humans.

Increase in body temperature has been thought to play an important role in the regulation of immune responses, although its precise mechanisms are still under investigation. Here, we examined the effects of physiologically relevant thermal stress on the cytokine production from human peripheral T cells. Volunteers were heated using a whole-body hyperthermia device, the rectal temperature was maintained above 38.5 °C for more than 60 min, and peripheral blood mononuclear cells (PBMCs) were obtained before and after the treatment. When T cells were stimulated with anti-CD3/CD28 antibodies, marked increases in the production of interferon-γ (IFN-γ) and interleukin-2 were observed in PBMCs prepared immediately after and 24h after the treatment. Similarly, enhanced production of IFN-γ in response to the tuberculin purified protein derivative or antigenic viral peptides was also observed immediately after and 24h after the treatment. Fluorescence photo-bleaching analyses showed heat-induced increase of membrane fluidity in T cells, which probably enables them to induce rapid and efficient cluster formation of molecules involved in antigen recognition and signal transduction for T-cell stimulation. We concluded that physiologically relevant thermal stress could efficiently modify T-cell responsiveness to various stimuli, including enhanced responses to specific antigens.

Development of a novel in vivo cancer model using cell sheet engineering.

AIM: Standard in vivo cancer models entail injecting single cancer cells, but this technique is not always successful. We developed a novel cancer cell sheet by using temperature-responsive polymer poly(N-isopropyl acryl amide)-coated plates, which allow controlled attachment and detachment of living cancer cells via simple temperature changes. MATERIALS AND METHODS: Four human cancer cell lines were used to make cell sheets. The cancer cell sheets were subcutaneously transplanted into nude mice and compared regarding their tumor-forming ability with the conventional cell suspension technique. RESULTS: Human cancer cell sheets were successfully transplanted into nude mice. The cancer cell sheets resulted in stable engraftment and showed a higher tumor volume determined by total flux with the IVIS® imaging system. CONCLUSION: Novel cancer cell sheets are useful tools to make in vivo cancer models in mice for the assessment of anticancer therapeutics.

[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.

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).

Final storage of radioactive cesium by pollucite hydrothermal synthesis.

The Fukushima nuclear accident has highlighted the importance of finding a better final storage method for radioactive cesium species. Cs is highly soluble in water, and can easily exchange with other alkali ions in zeolites or clays to form stable complexes. However, Cs(+) is released from Cs(+) complexes into water when surrounded by an excess of water. Pollucite may be the best final storage option for Cs(+), but its typical synthesis requires heating to about 1200 °C in air. Here, we show that the hydrothermal synthesis of pollucite can be completed at 300 °C in three hours from any zeolite or clay. Furthermore, our procedure does not require ion exchange before synthesis. Radioactive Cs is usually found in complexes with clays. At that time, this method only requires calcium hydroxide, water, and three hours of hydrothermal synthesis, so the process is both inexpensive and practical for large-scale application. Pollucite is an analog of analcime zeolite, and contains a channel system 2.8 Å in diameter, which is formed by 6-oxygen rings. As the diameter of Cs(+) is 3.34 Å and each Cs(+) exists independently within a separate portion of the channel, Cs(+) cannot exit the pollucite framework without breaking it.

Postoperative dendritic cell vaccine plus activated T-cell transfer improves the survival of patients with invasive hepatocellular carcinoma.

The recurrence rate after surgery in patients with hepatocellular carcinoma (HCC) is very high, while prognosis is quite poor. However, there is no standard treatment to prevent recurrence of HCC after a curative operation. In this study, we investigated the clinical utilization of an autologous tumor lysate-pulsed dendritic cell vaccine plus ex vivo activated T cell transfer (ATVAC) in an adjuvant setting for postoperative HCC as a non-randomized controlled trial. Ninety-four patients with invasive HCC received informed consent information regarding the study, and 42 opted to have the ATVAC after surgery. Their recurrence-free survival (RFS) and overall survival (OS) were measured after 5 years and compared with those of 52 patients who selected to have the curative operation alone. The median RFS and OS were 24.5 months and 97.7 months in the patients receiving adjuvant ATVAC and 12.6 months and 41.0 months in the group receiving surgery alone (P = 0.011 and 0.029). In the treated group, patients with positive delayed-type hypersensitivity (DTH) had a better prognosis (RFS P = 0.019, OS P = 0.025). No adverse events of grade 3 or more were observed. A postoperative dendritic cell vaccine plus activated T cell transfer would be a feasible and effective treatment for preventing recurrence in HCC patients and achieving long-term survival especially in DTH positive patients.

Global development strategy for companion diagnostics based on the usage and approval history for biomarkers in Japan, the USA and the EU.

AIM: The aim of this study was to identify gaps between Japan and the West in biomarker usage and the development of companion diagnostics. We also elaborated potential scenarios for companion diagnostic development. METHODS: Information on drug labels in Japan, the USA and the EU was obtained from each regulatory authority's web site, as well as label information on in vitro diagnostic testing in Japan and the USA. RESULTS: It is necessary to consider two factors when developing companion diagnostics globally: ethnic differences in gene mutations, and the approach to patient selection in clinical trials. A flowchart covering four scenarios was developed. CONCLUSION: Two factors that should be taken account when developing companion diagnostics globally was specified. This flowchart is expected to serve as a guide for streaming the development of companion diagnostics.