Challenges and gaps in immunosafety evaluation of therapeutics: An IQ DruSafe survey.

Immunotoxicology/immunosafety science is rapidly evolving, with novel modalities and immuno-oncology among the primary drivers of new tools and technologies. The Immunosafety Working Group of IQ/DruSafe sought to better understand some of the key challenges in immunosafety evaluation, gaps in the science, and current limitations in methods and data interpretation. A survey was developed to provide a baseline understanding of the needs and challenges faced in immunosafety assessments, the tools currently being applied across the industry, and the impact of feedback received from regulatory agencies. This survey also focused on current practices and challenges in conducting the T-cell-dependent antibody response (TDAR) and the cytokine release assay (CRA). Respondents indicated that ICH S8 guidance was insufficient for the current needs of the industry portfolio of immunomodulators and novel modalities and should be updated. Other challenges/gaps identified included translation of nonclinical immunosafety assessments to the clinic, and lack of relevant nonclinical species and models in some cases. Key areas of emerging science that will add future value to immunotoxicity assessments include development of additional in vitro and microphysiological system models, as well as application of humanized mouse models. Efforts are ongoing in individual companies and consortia to address some of these gaps and emerging science.

Current nonclinical approaches for immune assessments of immuno-oncology biotherapeutics.

Harnessing the immune system to kill tumors has been revolutionary and, as a result, has had an enormous benefit for patients in extending life and resulting in effective cures in some. However, activation of the immune system can come at the cost of undesirable adverse events such as cytokine release syndrome, immune-related adverse events, on-target/off-tumor toxicity, neurotoxicity and tumor lysis syndrome, which are safety risks that can be challenging to assess non-clinically. This article provides a review of the biology and mechanisms that can result in immune-mediated adverse effects and describes industry approaches using in vitro and in vivo models to aid in the nonclinical safety risk assessments for immune-oncology modalities. Challenges and limitations of knowledge and models are also discussed.

Mutation of the phospholipase C-gamma1-binding site of LAT affects both positive and negative thymocyte selection.

Linker for activation of T cells (LAT) is a scaffolding adaptor protein that is critical for T cell development and function. A mutation of LAT (Y136F) that disrupts phospholipase C-gamma1 activation and subsequent calcium influx causes a partial block in T cell development and leads to a severe lymphoproliferative disease in homozygous knock-in mice. One possible contribution to the fatal disease of LAT Y136F knock-in mice could be from autoreactive T cells generated in these mice because of altered thymocyte selection. To examine the impact of the LAT Y136F mutation on thymocyte positive and negative selection, we bred this mutation onto the HY T cell receptor (TCR) transgenic, recombination activating gene-2 knockout background. Female mice with this genotype showed a severe defect in positive selection, whereas male mice exhibited a phenotype resembling positive selection (i.e., development and survival of CD8(hi) HY TCR-specific T cells) instead of negative selection. These results support the hypothesis that in non-TCR transgenic, LAT Y136F knock-in mice, altered thymocyte selection leads to the survival and proliferation of autoreactive T cells that would otherwise be negatively selected in the thymus.

Nonclinical Studies that Support Viral Vector-Delivered Gene Therapies: An EFPIA Gene Therapy Working Group Perspective.

Nonclinical development strategies for gene therapies are unique from other modalities. The European Federation of Pharmaceutical Industries and Associates (EFPIA) Gene Therapy Working Group surveyed EFPIA member and nonmember pharmaceutical and biotechnology companies about their current practices for designing and implementing nonclinical toxicology studies to support the development of viral vector-delivered in vivo gene therapies. Compiled responses from 17 companies indicated that these studies had some variability in species selection, study-design elements, biodistribution, immunogenicity or genomic insertion assessments, safety pharmacology, and regulatory interactions. Although there was some consistency in general practice, there were examples of extreme case-by-case differences. The responses and variability are discussed herein. Key development challenges were also identified. Results from this survey emphasize the importance for harmonization of regulatory guidelines for the development of gene-therapy products, while still allowing for case-by-case flexibility in nonclinical toxicology studies. However, the appropriate timing for a harmonized guidance, particularly with a platform that continues to rapidly evolve, remains in question.

Transcriptome analysis of human immune responses following live vaccine strain (LVS) Francisella tularensis vaccination.

The live vaccine strain (LVS) of Francisella tularensis is the only vaccine against tularemia available for humans, yet its mechanism of protection remains unclear. We probed human immunological responses to LVS vaccination with transcriptome analysis using PBMC samples from volunteers at time points pre- and post-vaccination. Gene modulation was highly uniform across all time points, implying commonality of vaccine responses. Principal components analysis revealed three highly distinct principal groupings: pre-vaccination (-144 h), early (+18 and +48 h), and late post-vaccination (+192 and +336 h). The most significant changes in gene expression occurred at early post-vaccination time points (

High-resolution multicolor imaging of dynamic signaling complexes in T cells stimulated by planar substrates.

The dynamic visualization of developing immunological synapses has been hindered by the difficulty of imaging the contact between the T cell and the antigen-presenting cell (APC). Here, we describe a technique in which T cell responses are constrained to a planar stimulatory substrate. This approach, when used in conjunction with immunofluorescent staining procedures or fluorescent protein tags, greatly facilitates detection of the dynamic molecular rearrangements that accompany the formation of contacts and the initiation of signal transduction through the T cell receptor (TCR). Using this method, we have observed signaling complexes of dynamically varying compositions that possess distinct fates.

Suppressed clinical experimental autoimmune myasthenia gravis in bm12 mice is linked to reduced intracellular calcium mobilization and IL-10 and IFN-gamma release by acetylcholine receptor-specific T cells.

Class II MHC mutant bm12 mice have an increased resistance to experimental autoimmune myasthenia gravis (EAMG) compared to C57BL/6 mice. In vitro, this relative resistance was mainly associated with a reduced cytokine response to acetylcholine receptor (AChR) and its dominant pathogenic peptide alpha 146-162, whereas the response to the epitope alpha 111-126 remained intact. Calcium mobilization after stimulation of AChR-immune T cells with AChR or alpha 146-162 peptide, but not alpha 111-126 peptide, was decreased in bm12 compared to C57BL/6. Thus, the reduced incidence of clinical EAMG in bm12 is linked to lower IFN-gamma and IL-10 release, and intracellular calcium mobilization by alpha 146-162-specific T cells.

NKp30-dependent cytolysis of filovirus-infected human dendritic cells.

Understanding how protective innate immune responses are generated is crucial to defeating highly lethal emerging pathogens. Accumulating evidence suggests that potent innate immune responses are tightly linked to control of Ebola and Marburg filoviral infections. Here, we report that unlike authentic or inactivated Ebola and Marburg, filovirus-derived virus-like particles directly activated human natural killer (NK) cells in vitro, evidenced by pro-inflammatory cytokine production and enhanced cytolysis of permissive target cells. Further, we observed perforin- and CD95L-mediated cytolysis of filovirus-infected human dendritic cells (DCs), primary targets of filovirus infection, by autologous NK cells. Gene expression knock-down studies directly linked NK cell lysis of infected DCs to upregulation of the natural cytotoxicity receptor, NKp30. These results are the first to propose a role for NK cells in the clearance of infected DCs and the potential involvement of NKp30-mediated cytolysis in control of viral infection in vivo. Further elucidation of the biology of NK cell activation, specifically natural cytotoxicity receptors like NKp30 and NKp46, promises to aid our understanding of microbial pathology.

Activation of triggering receptor expressed on myeloid cells-1 on human neutrophils by marburg and ebola viruses.

Marburg virus (MARV) and Ebola virus (EBOV), members of the viral family Filoviridae, cause fatal hemorrhagic fevers in humans and nonhuman primates. High viral burden is coincident with inadequate adaptive immune responses and robust inflammatory responses, and virus-mediated dysregulation of early host defenses has been proposed. Recently, a novel class of innate receptors called the triggering receptors expressed in myeloid cells (TREM) has been discovered and shown to play an important role in innate inflammatory responses and sepsis. Here, we report that MARV and EBOV activate TREM-1 on human neutrophils, resulting in DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory cytokines, and phenotypic changes. A peptide specific to TREM-1 diminished the release of tumor necrosis factor alpha by filovirus-activated human neutrophils in vitro, and a soluble recombinant TREM-1 competitively inhibited the loss of cell surface TREM-1 that otherwise occurred on neutrophils exposed to filoviruses. These data imply direct activation of TREM-1 by filoviruses and also indicate that neutrophils may play a prominent role in the immune and inflammatory responses to filovirus infections.

Dendritic cells endocytose Bacillus anthracis spores: implications for anthrax pathogenesis.

Phagocytosis of inhaled Bacillus anthracis spores and subsequent trafficking to lymph nodes are decisive events in the progression of inhalational anthrax because they initiate germination and dissemination of spores. Found in high frequency throughout the respiratory track, dendritic cells (DCs) routinely take up foreign particles and migrate to lymph nodes. However, the participation of DCs in phagocytosis and dissemination of spores has not been investigated previously. We found that human DCs readily engulfed fully pathogenic Ames and attenuated B. anthracis spores predominately by coiling phagocytosis. Spores provoked a loss of tissue-retaining chemokine receptors (CCR2, CCR5) with a concurrent increase in lymph node homing receptors (CCR7, CD11c) on the membrane of DCs. After spore infection, immature DCs displayed a mature phenotype (CD83(bright), HLA-DR(bright), CD80(bright), CD86(bright), CD40(bright)) and enhanced costimulatory activity. Surprisingly, spores activated the MAPK cascade (ERK, p38) within 30 min and stimulated expression of several inflammatory response genes by 2 h. MAPK signaling was extinguished by 6 h infection, and there was a dramatic reduction of secreted TNF-alpha, IL-6, and IL-8 in the absence of DC death. This corresponded temporally with enzymatic cleavage of proximal MAPK signaling proteins (MEK-1, MEK-3, and MAP kinase kinase-4) and may indicate activity of anthrax lethal toxin. Taken together, these results suggest that B. anthracis may exploit DCs to facilitate infection.

All roads lead to actin: the intimate relationship between TCR signaling and the cytoskeleton.

Regardless of cell type, the regulation of the actin cytoskeleton is tightly linked to vital biological properties such as polarity, motility, cell-cell contact, exocytosis and proliferation. In the immune system, where rapid and efficient response to antigen-provoked stimuli is crucial, an overwhelming amount of data implicate the actin cytoskeleton and its regulators as central to immune function. Increasingly, the cytoskeleton is considered an essential amplification step in T cell receptor (TCR)-, costimulatory-, and integrin-mediated signaling. Advances in genetic manipulation and confocal imaging have led to a keener appreciation of the importance of TCR signal integration by the actin cytoskeleton. This review outlines recent advances in elucidating the regulation of T cell function through the actin cytoskeleton. We also examine intriguing parallels between the immune system and other models of cytoskeletal regulation.

A LAT mutation that inhibits T cell development yet induces lymphoproliferation.

Mice homozygous for a single tyrosine mutation in LAT (linker for activation of T cells) exhibited an early block in T cell maturation but later developed a polyclonal lymphoproliferative disorder and signs of autoimmune disease. T cell antigen receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1) and of nuclear factor of activated T cells, calcium influx, interleukin-2 production, and cell death were reduced or abrogated in T cells from LAT mutant mice. In contrast, TCR-induced Erk activation was intact. These results identify a critical role for integrated PLC-gamma1 and Ras-Erk signaling through LAT in T cell development and homeostasis.