Alternatives to Monkey Reproductive Toxicology Testing for Biotherapeutics.

Embryofetal toxicity studies are conducted to support inclusion of women of childbearing potential in clinical trials and to support labeling for the marketed pharmaceutical product. For biopharmaceuticals, which frequently lack activity in the rodent or rabbit, the nonhuman primate is the standard model to evaluate embryofetal toxicity. These studies have become increasingly challenging to conduct due to the small number of facilities capable of performing them and a shortage of sexually mature monkeys. The low number of animals per group and the high rate of spontaneous abortion in cynomolgus monkeys further complicate interpretation of the data. Recent FDA guidance has proposed a weight of evidence (WoE) approach to support product labeling for reproductive toxicity of products intended to be used for the treatment of cancer (Oncology Pharmaceuticals: Reproductive Toxicity Testing and Labeling Recommendations), an approach that has also supported the approval of biotherapeutics for non-cancer indications. Considerations to determine the appropriateness and content of a WoE approach to support product labeling for embryofetal risk include known class effects in humans; findings from genetically modified animals with or without drug administration; information from surrogate compounds; literature-based assessments about the developmental role of the pharmaceutical target; and the anticipated exposure during embryofetal development. This paper summarizes the content of a session presented at the 42nd annual meeting at the American College of Toxicology, which explored the conditions under which alternative approaches may be appropriate to support product labeling for reproductive risk, and how sponsors can best justify the use of this approach.

Addressing practical issues in the smooth implementation of revised guidelines for non-clinical studies of vaccines for infectious disease prevention.

Herein, we investigated possible practical issues for the smooth implementation of the revised Japanese Guidelines for Non-clinical Studies of Vaccines for the Prevention of Infectious Diseases, which were raised in response to public comments on the proposed guideline revision and a gap analysis of the World Health Organization and European Medicines Agency guidelines. We identified main issues such as the non-clinical safety studies of adjuvants and evaluation of local cumulative tolerance in toxicity studies. The revised Japanese Pharmaceuticals and Medical Devices Agency (PMDA)/Ministry of Health, Labour and Welfare (MHLW) guidelines require non-clinical safety studies for vaccines containing new adjuvants, but additional safety pharmacology studies or safety studies in two animal species may be required if non-clinical safety studies raise any concerns (i.e., systemic distribution). Adjuvant biodistribution studies may aid in understanding vaccine characteristics. The evaluation of local cumulative tolerance in non-clinical studies, which was the focus of the Japanese review, can be omitted by including a warning in the package insert to avoid injection to the same site. The study's findings will be reflected in a Q&A to be released by the Japanese MHLW. We hope that this study will contribute to the global and harmonized development of vaccines.

Proposal for the revision of the guidelines for Non-clinical studies of vaccines for the prevention of infectious diseases in Japan.

The efficacy and safety of vaccines for the prevention of infectious diseases are mostly evaluated based on the induction of an immune response against antigens, and do not necessarily depend on the dose administered. Therefore, there are some specific aspects that need to be considered in the development of vaccines and have been described in "The Guidelines for the non-clinical studies of vaccines for the prevention of infectious disease" in Japan. Recent changes in the vaccine development field, such as the introduction of vaccines developed overseas in Japan and vaccine development on a global scale have increased the need for revision of these guidelines. In this study, we identified the current challenges in the development of vaccines through comparison of Japanese and international guidelines. We conducted a questionnaire-based survey of pharmaceutical industries in Japan, and found issues related to non-clinical studies, such as the necessity of safety pharmacology studies and repeated-dose toxicity studies for each route of administration. We examined international guidelines on these issues as well as review reports by regulatory authorities, and determined that the results of repeated-dose toxicity studies can be used to decide whether safety pharmacology studies are required, and that studies to evaluate toxicity due to systemic effects may not be necessary for both intramuscular and subcutaneous administration. We propose revision of the guidelines for the non-clinical studies of vaccines in Japan taking international harmonizaion into account. We expected that the revised guidelines will promote smooth and rational vaccine development.

Nonclinical safety assessment of engineered T cell therapies.

Over the last decade, immunotherapy has established itself as an important novel approach in the treatment of cancer, resulting in a growing importance in oncology. Engineered T cell therapies, namely chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) T cell therapies, are platform technologies that have enabled the development of products with remarkable efficacy in several hematological malignancies and are thus the focus of intense research and development activity. While engineered T cell therapies offer promise in addressing currently intractable cancers, they also present unique challenges, including their nonclinical safety assessment. A workshop organized by HESI and the US Food and Drug Administration (FDA) was held to provide an interdisciplinary forum for representatives of industry, academia and regulatory authorities to share information and debate on current practices for the nonclinical safety evaluation of engineered T cell therapies. This manuscript leverages what was discussed at this workshop to provide an overview of the current important nonclinical safety assessment considerations for the development of these therapeutic modalities (cytokine release syndrome, neurotoxicity, on-target/off-tumor toxicities, off-target effects, gene editing or vector integration-associated genomic injury). The manuscript also discusses approaches used for hazard identification or risk assessment and provides a regulatory perspective on such aspects.

Considerations of the Japanese Research Working Group for the ICH S6 & Related Issues Regarding Nonclinical Safety Assessments of Oligonucleotide Therapeutics: Comparison with Those of Biopharmaceuticals.

This white paper summarizes the current consensus of the Japanese Research Working Group for the ICH S6 & Related Issues (WGS6) on strategies for the nonclinical safety assessment of oligonucleotide-based therapeutics (ONTs), specifically focused on the similarities and differences to biotechnology-derived pharmaceuticals (biopharmaceuticals). ONTs, like biopharmaceuticals, have high species and target specificities. However, ONTs have characteristic off-target effects that clearly differ from those of biopharmaceuticals. The product characteristics of ONTs necessitate specific considerations when planning nonclinical studies. Some ONTs have been approved for human use and many are currently undergoing nonclinical and/or clinical development. However, as ONTs are a rapidly evolving class of drugs, there is still much to learn to achieve optimal strategies for the development of ONTs. There are no formal specific guidelines, so safety assessments of ONTs are principally conducted by referring to published white papers and conventional guidelines for biopharmaceuticals and new chemical entities, and each ONT is assessed on a case-by-case basis. The WGS6 expects that this report will be useful in considering nonclinical safety assessments and developing appropriate guidelines specific for ONTs.

Aspects of Gene Therapy Products Using Current Genome-Editing Technology in Japan.

The development of genome-editing technology could lead to breakthrough gene therapy. Genome editing has made it possible to easily knock out or modify a target gene, while current gene therapy using a virus vector or plasmid hampering modification with respect to gene replacement therapies. Clinical development using these genome-editing tools is progressing rapidly. However, it is also becoming clear that there is a possibility of unintended gene sequence modification or deletion, or the insertion of undesired genes, or the selection of cells with abnormalities in the cancer suppressor gene p53; these unwanted actions are not possible with current gene therapy. The Science Board of the Pharmaceuticals and Medical Devices Agency of Japan has compiled a report on the expected aspects of such genome-editing technology and the risks associated with it. This article summarizes the history of that discussion and compares the key concepts with information provided by other regulatory authorities.

Investigating toxicity specific to adjuvanted vaccines.

In an attempt to understand the unique toxicity of adjuvanted vaccines, we studied how toxicity develops over time following vaccine administration. In addition to on- and off-target toxicity typically observed with general pharmaceuticals, we observed toxicity associated with both the generation and the broad action of effectors (antibodies and/or cytotoxic T lymphocytes, CTLs). The impact on effector generation appears to be related to local tolerance specific to the adjuvant. The vaccine immune response by effectors serves to demonstrate species relevance as outlined in the recent WHO guideline on the nonclinical evaluation of adjuvanted vaccines. When regarded as pharmaceuticals that function at sites of local administration, adjuvants have inherent on- and off-target toxicity. On-target toxicity of the adjuvant is typically associated with effector generation, and could vary depending on animal species. Therefore, the use of species with sensitivity to adjuvants described in the WHO guidelines is required to evaluate the toxicity of the vaccine associated with effector generation. Changes in safety pharmacology endpoints would be considered off-target and further studies are conducted only if changes in these endpoints are observed in nonclinical or clinical studies. Thus our decision tree does not recommend the routine conduct of stand-alone safety pharmacology studies.

Considerations for non-clinical safety studies of therapeutic peptide vaccines.

Guidelines for non-clinical studies of prophylactic vaccines against infectious diseases have been published widely, but similar guidelines for therapeutic vaccines, and especially therapeutic peptide vaccines, have yet to be established. The approach to non-clinical safety studies required for therapeutic vaccines differs from that for prophylactic vaccines due to differences in the risk-benefit balance and the mechanisms of action. We propose the following guidelines for non-clinical safety studies for therapeutic peptide vaccines. (i) Since the main safety concern is related to the immune response that might occur at normal sites that express a target antigen, identification of these possible target sites using in silico human expression data is important. (ii) Due to the strong dependence on HLA, it is not feasible to replicate immune responses in animals. Thus, the required non-clinical safety studies are characterized as those detecting off-target toxicity rather than on-target toxicity.

Role of hepatocyte-derived IL-7 in maintenance of intrahepatic NKT cells and T cells and development of B cells in fetal liver.

The liver contains a variety of resident immune cells, such as NK cells, NKT cells, T cells, macrophages, and dendritic cells. However, little is known about how IL-7, which is produced by hepatocytes, functions locally in development and maintenance of liver immune cells. To address this question, we established IL-7-floxed mice and crossed them with albumin promoter-driven Cre (Alb-Cre) transgenic mice to establish conditional knockout of IL-7 in hepatocytes. The levels of IL-7 transcripts were reduced 10-fold in hepatocyte fraction. We found that the absolute numbers of NKT and T cells were significantly decreased in adult liver of IL-7(f/f) Alb-Cre mice compared with IL-7(f/f) control mice. In contrast, NK cells, dendritic cells, and B cells were unchanged in the IL-7(f/f) Alb-Cre liver. The number of Vα14(+) invariant NKT cells was significantly reduced in liver, but not in thymus and spleen, of IL-7(f/f) Alb-Cre mice. Furthermore, B cell development was impaired in perinatal liver of IL-7(f/f) Alb-Cre mice. This study demonstrates that hepatocyte-derived IL-7 plays an indispensable role in maintenance of NKT and T cells in adult liver and development of B cells in fetal liver, and suggests that hepatocytes provide a unique IL-7 niche for intrahepatic lymphocytes.

MEK1/2 induces STAT5-mediated germline transcription of the TCRgamma locus in response to IL-7R signaling.

The IL-7R plays an essential role in gammadelta T cell development by inducing V-J recombination of the TCRgamma locus through STAT5. Although tyrosine residues in the intracellular domain of the mouse IL-7R alpha-chain (IL-7Ralpha) have been implicated in STAT5 activation, it is still unknown whether they are essential for gammadelta T cell development. In this study, we showed that those IL-7Ralpha tyrosine residues are not essential for gammadelta T cell development, because phenylalanine replacement of four intracellular tyrosine residues (IL-7R-FFFF) partially rescued gammadelta T cell development of IL-7Ralpha-/- progenitors. To examine signaling pathways activated by IL-7R-FFFF, we introduced a chimeric receptor consisting of the human IL-4R alpha-chain and mouse IL-7R-FFFF (4R/7R-FFFF) into an IL-7-dependent pre-B cell line and found that 4R/7R-FFFF induced TCRgamma germline transcription and STAT5 activation. Treatment of cells with MEK1/2 inhibitors significantly decreased levels of TCRgamma germline transcription and STAT5 tyrosine phosphorylation mediated by 4R/7R-FFFF, suggesting that MEK1/2 plays an alternative role in STAT5 activation by IL-7R. MEK1/2 associated with STAT5 and induced STAT5 tyrosine phosphorylation and DNA binding activity. Furthermore, MEK1 directly phosphorylated a STAT5 tyrosine residue in vitro. Finally, active MEK1 partially rescued TCRgamma germline transcription by IL-7R in a pre-T cell line. These results demonstrate that MEK1/2 induces TCRgamma germline transcription by phosphorylating STAT5 through IL-7R-FFFF and suggest a potential role for MAPK in IL-7R tyrosine-independent activation of STAT5.

Transcriptional activation of mouse TCR Jgamma4 germline promoter by STAT5.

The IL-7 receptor (IL-7R) controls the accessibility of mouse TCRgamma locus, by recruiting STAT5 and transcriptional coactivators to the Jgamma1 germline promoter and inducing histone acetylation at nearby chromatin. Although a STAT motif is present in Jgamma4 germline promoter, it is still unknown whether STAT5 regulates the transcription of the Jgamma4 promoter. Here, we showed that cytokine stimulation induced Jgamma4-Cgamma4 germline transcripts in a pre-T cell line, Scid.adh, and a hematopoietic cell line, Ba/F3. A STAT consensus motif was present in 5' region of Jgamma4 gene segment. We found that STAT5 bound to the STAT motif of the Jgamma4 germline promoter in vitro by EMSA. In addition, we detected by chromatin immunoprecipitation assay that STAT5 was recruited to the endogenous Jgamma4 chromatin in Ba/F3 cells after cytokine stimulation. Finally, using reporter assay, we showed that the Jgamma4 germline promoter was activated by STAT5 and that mutation in the STAT motif abrogated the activity. Furthermore, this transactivation was augmented by transcriptional coactivators, CBP and p300. Collectively, these results demonstrate that STAT5 binds to the STAT motif in the Jgamma4 promoter and induces germline transcription. Thus, this study indicates that the IL-7R/STAT5 signal controls the transcription and accessibility of different clusters in the TCRgamma locus.

Induction of the IL-7 receptor alpha chain in mouse peripheral B cells by glucocorticoids.

Expression of the IL-7R alpha chain (IL-7R alpha) is strictly regulated during development and maturation of lymphocytes. While T cells express the IL-7R alpha in the periphery, B cells do not. Glucocorticoids (GCs) have pleiotypic effects on development and function of lymphocytes. Although GCs induce the transcription of IL-7R alpha gene in T cells, their effect on B cells is largely unknown. Here, we show that GCs induce the transcription and expression of IL-7R alpha in mouse peripheral B cells. This effect does not require de novo protein synthesis, because a protein synthesis inhibitor, cycloheximide, does not block the transcription. IL-7R signal pathway is intact in peripheral B cells because Stat5, one of the signal molecules of the IL-7R alpha, is phosphorylated by IL-7 stimulation. We also observed that IL-7 simulation induces the transcription of Cis-1, one of the target genes of Stat5. Furthermore, GC-induced IL-7R alpha can transmit survival signal in B cells. Therefore, this study demonstrates that GCs induce the transcription and expression of functional IL-7R alpha in peripheral B cells, and implies a potential role of the IL-7R in survival of B cells.

Transcriptional regulation of the mouse IL-7 receptor alpha promoter by glucocorticoid receptor.

Expression of the IL-7R alpha-chain (IL-7Ralpha) is strictly regulated during the development and maturation of lymphocytes. Glucocorticoids (GC) have pleiotypic effects on the growth and function of lymphocytes. Although GC have been reported to induce the transcription of IL-7Ralpha gene in human T cells, its molecular mechanism is largely unknown. In this study, we show that GC up-regulate the levels of IL-7Ralpha mRNA and protein in mouse T cells. This effect does not require protein synthesis de novo, because protein synthesis inhibitors do not block the process. Mouse IL-7Ralpha promoter has striking homology with human and rat, containing consensus motifs of Ikaros, PU.1, and Runx1 transcription factors. In addition, a conserved noncoding sequence (CNS) of approximately 270 bp was found 3.6-kb upstream of the promoter, which was designated as CNS-1. A GC receptor (GR) motif is present in the CNS-1 region. Importantly, we show by reporter assay that the IL-7Ralpha promoter has specific transcription activity in T cells. This activity highly depends on the PU.1 motif. Furthermore, GC treatment augments the transcriptional activity through the GR motif in the CNS-1 region. We also demonstrate that GR binds to the GR motif by EMSA. In addition, by chromatin immunoprecipitation assay, we show that GR is rapidly recruited to endogenous CNS-1 chromatin after GC stimulation. These results demonstrate that GR binds to the GR motif in the CNS-1 region after GC stimulation and then activates the transcription of the IL-7Ralpha promoter. Thus, this study identifies the IL-7Ralpha CNS-1 region as a GC-responsive element.

Phosphorylation controls Ikaros's ability to negatively regulate the G(1)-S transition.

Ikaros is a key regulator of lymphocyte proliferative responses. Inactivating mutations in Ikaros cause antigen-mediated lymphocyte hyperproliferation and the rapid development of leukemia and lymphoma. Here we show that Ikaros's ability to negatively regulate the G(1)-S transition can be modulated by phosphorylation of a serine/threonine-rich conserved region (p1) in exon 8. Ikaros phosphorylation in p1 is induced during the G(1)-S transition. Mutations that prevent phosphorylation in p1 increase Ikaros's ability to impede cell cycle progression and its affinity for DNA. Casein kinase II, whose increased activity in lymphocytes leads to transformation, is a key player in Ikaros p1 phosphorylation. We thus propose that Ikaros's activity as a regulator of the G(1)-S transition is controlled by phosphorylation in response to signaling events that down-modulate its DNA binding activity.

Molecular cloning and characterization of TPP36 and its isoform TPP32, novel substrates of Abl tyrosine kinase.

We have molecularly cloned TPP36, a novel 36 kDa protein with 281 amino acids that was identified as a protein phosphorylated in B progenitor cells following stimulation with pervanadate/H(2)O(2). Analysis with anti-TPP36 antiserum revealed that TPP36 was expressed ubiquitously and had an isoform with 236 amino acids, designated TPP32. TPP36/32 were localized mainly in cytoplasm despite the presence of a typical nuclear localization signal sequence. These proteins were phosphorylated preferentially by Abl among a panel of tyrosine kinases examined. Phosphorylation of tyrosine 120 in TPP36/32 led to an apparent mobility shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting conformational change in the phosphorylated protein. Thus, TPP36/32 appear to be novel substrates of Abl tyrosine kinase.