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.

Nonclinical pharmacokinetics and biodistribution of VSV-GP using methods to decouple input drug disposition and viral replication.

Viral replication places oncolytic viruses (OVs) in a unique niche in the field of drug pharmacokinetics (PK) as their self-amplification obscures exposure-response relationships. Moreover, standard bioanalytical techniques are unable to distinguish the input from replicated drug products. Here, we combine two novel approaches to characterize PK and biodistribution (BD) after systemic administration of vesicular stomatitis virus pseudotyped with lymphocytic choriomeningitis virus glycoprotein (VSV-GP) in healthy mice. First: to decouple input drug PK/BD versus replication PK/BD, we developed and fully characterized a replication-incompetent tool virus that retained all other critical attributes of the drug. We used this approach to quantify replication in blood and tissues and to determine its impact on PK and BD. Second: to discriminate the genomic and antigenomic viral RNA strands contributing to replication dynamics in tissues, we developed an in situ hybridization method using strand-specific probes and assessed their spatiotemporal distribution in tissues. This latter approach demonstrated that distribution, transcription, and replication localized to tissue-resident macrophages, indicating their role in PK and BD. Ultimately, our study results in a refined PK/BD profile for a replicating OV, new proposed PK parameters, and deeper understanding of OV PK/BD using unique approaches that could be applied to other replicating vectors.

Immunocompetent cancer-on-chip models to assess immuno-oncology therapy.

The advances in cancer immunotherapy come with several obstacles, limiting its widespread use and benefits so far only to a small subset of patients. One of the underlying challenges remains to be the lack of representative nonclinical models that translate to human immunity and are able to predict clinical efficacy and safety outcomes. In recent years, immunocompetent Cancer-on-Chip models emerge as an alternative human-based platform that enables the integration and manipulation of complex tumor microenvironment. In this review, we discuss novel opportunities offered by Cancer-on-Chip models to advance (mechanistic) immuno-oncology research, ranging from design flexibility to multimodal analysis approaches. We then exemplify their (potential) applications for the research and development of adoptive cell therapy, immune checkpoint therapy, cytokine therapy, oncolytic virus, and cancer vaccines.

Industry experiences with immune-mediated findings in biotherapeutic nonclinical toxicology studies.

With the growth of monoclonal antibodies and other proteins as major modalities in the pharmaceutical industry, there has been an increase in pharmacology and toxicity testing of biotherapeutics in animals. Animals frequently mount an immune response to human therapeutic proteins. This can result in asymptomatic anti-drug antibody formation, immune complexes that affect drug disposition and/or organ function such as kidney, cytokine release responses, fatal hypersensitivity, or a range of reactions in between. In addition, an increasing number of oncology therapeutics are being developed that enhance or directly stimulate immune responses by a variety of mechanisms, which could increase the risk of autoreactivity and an autoimmune-like syndrome in animals and humans. When evaluating the risk of biotherapeutics prior to entering the clinic, the nonclinical safety data may include any of these responses and it is critical to understand whether they represent a safety liability for humans. The DruSafe Leadership group of the IQ Consortium conducted a survey of industry to understand sponsors' experiences with these immune reactions in nonclinical studies related to both immunogenicity and pharmacologically-mediated immune perturbations. The survey covered what pathways were affected, how the immune responses were presented, how the company and health authorities interpreted the data and whether the immune responses were observed in the clinic. Additionally, the survey gathered information on association of these findings with anti-drug antibodies as well as sponsor's use of immunogenicity predictive tools. The data suggests that the ability of a biotherapeutic to activate the immune system, intended or not, plays a significant role on characteristics of the response and whether theys are translatable.

VHH antibody targeting the chemokine receptor CX3CR1 inhibits progression of atherosclerosis.

CX3CR1 has been identified as a highly attractive target for several therapeutic interventions. Despite this potential, no potent antagonists, either small molecule or monoclonal antibody, have been identified. Here we describe the lead finding and engineering approach that lead to the identification of BI 655088, a potent biotherapeutic antagonist to CX3CR1. BI 655088 is a potent CX3CR1 antagonist that, upon therapeutic dosing, significantly inhibits plaque progression in the standard mouse model of atherosclerosis. BI 655088 represents a novel and highly selective biotherapeutic that could reduce inflammation in the atherosclerotic plaque when added to standard of care treatment including statins, which could result in a significant decrease in atherothrombotic events in patients with existing cardiovascular disease.

Reperfusion injury intensifies the adaptive human T cell alloresponse in a human-mouse chimeric artery model.

OBJECTIVE: Perioperative nonimmune injuries to an allograft can decrease graft survival. We have developed a model for studying this process using human materials. METHODS AND RESULTS: Human artery segments were transplanted as infrarenal aortic interposition grafts into an immunodeficient mouse host, allowed to "heal in" for 30 days, and then retransplanted into a second mouse host. To induce a reperfusion injury, the healed-in artery segments were incubated for 3 hours under hypoxic conditions ex vivo before retransplantation. To induce immunologic rejection, the animals receiving the retransplanted artery segment were adoptively transferred with human peripheral blood mononuclear cells or purified T cells from a donor allogeneic to the artery 1 week before surgery. To compare rejection of injured versus healthy tissues, these manipulations were combined. Results were analyzed ex vivo by histology, morphometry, immunohistochemistry, and mRNA quantitation or in vivo by ultrasound. Our results showed that reperfusion injury, which otherwise heals with minimal sequelae, intensifies the degree of allogeneic T cell-mediated injury to human artery segments. CONCLUSIONS: We developed a new human-mouse chimeric model demonstrating interactions of reperfusion injury and alloimmunity using human cells and tissues that may be adapted to study other forms of nonimmune injury and other types of adaptive immune responses.

Neutralizing IL-6 reduces human arterial allograft rejection by allowing emergence of CD161+ CD4+ regulatory T cells.

Perioperative injuries to an allograft exacerbate graft rejection, which in humans is primarily mediated by effector memory T cells. IL-6 transcripts in human coronary artery segments rapidly increase posttransplantation into immunodeficient mouse hosts compared with those of pretransplant specimens and fall dramatically by 30 d. Adoptive transfer of human PBMCs allogeneic to the artery 2 d postoperatively results in T cell infiltrates and intimal expansion 4 wk later. Ab neutralization of human IL-6 reduces the magnitude of intimal expansion and total T cell infiltration but increases the relative expression of CD161 while decreasing other Th17 markers. Coculture of MHC class II-expressing human endothelial cells (ECs) with allogeneic CD4(+) memory T cells results in T cell activation and EC secretion of IL-6. Neutralizing IL-6 in primary allogeneic T cell-EC cocultures results in enhanced T cell proliferation of CD161(+) CD4(+) T cells, reduces total T cell proliferation upon restimulation in secondary cultures (an effect dependent on CD161(+) T cells), increases expression of FOXP3 in CD161(+) T cells, and generates T cells that suppress proliferation of freshly isolated T cells. These data suggest that IL-6 released from injured allograft vessels enhances allogeneic T cell infiltration and intimal expansion in a model of human allograft rejection by inhibiting an increase in CD161(+) regulatory T cells.

Diazoxide promotes oligodendrocyte precursor cell proliferation and myelination.

BACKGROUND: Several clinical conditions are associated with white matter injury, including periventricular white matter injury (PWMI), which is a form of brain injury sustained by preterm infants. It has been suggested that white matter injury in this condition is due to altered oligodendrocyte (OL) development or death, resulting in OL loss and hypomyelination. At present drugs are not available that stimulate OL proliferation and promote myelination. Evidence suggests that depolarizing stimuli reduces OL proliferation and differentiation, whereas agents that hyperpolarize OLs stimulate OL proliferation and differentiation. Considering that the drug diazoxide activates K(ATP) channels to hyperpolarize cells, we tested if this compound could influence OL proliferation and myelination. METHODOLOGY/FINDINGS: Studies were performed using rat oligodendrocyte precursor cell (OPC) cultures, cerebellar slice cultures, and an in vivo model of PWMI in which newborn mice were exposed to chronic sublethal hypoxia (10% O(2)). We found that K(ATP) channel components Kir 6.1 and 6.2 and SUR2 were expressed in oligodendrocytes. Additionally, diazoxide potently stimulated OPC proliferation, as did other K(ATP) activators. Diazoxide also stimulated myelination in cerebellar slice cultures. We also found that diazoxide prevented hypomyelination and ventriculomegaly following chronic sublethal hypoxia. CONCLUSIONS: These results identify KATP channel components in OLs and show that diazoxide can stimulate OL proliferation in vitro. Importantly we find that diazoxide can promote myelination in vivo and prevent hypoxia-induced PWMI.

Interleukin-1beta: a bridge between inflammation and excitotoxicity?

Interleukin-1 (IL-1) is a proinflammatory cytokine released by many cell types that acts in both an autocrine and/or paracrine fashion. While IL-1 is best described as an important mediator of the peripheral immune response during infection and inflammation, increasing evidence implicates IL-1 signaling in the pathogenesis of several neurological disorders. The biochemical pathway(s) by which this cytokine contributes to brain injury remain(s) largely unidentified. Herein, we review the evidence that demonstrates the contribution of IL-1beta to the pathogenesis of both acute and chronic neurological disorders. Further, we highlight data that leads us to propose IL-1beta as the missing mechanistic link between a potential beneficial inflammatory response and detrimental glutamate excitotoxicity.

System x(c)- activity and astrocytes are necessary for interleukin-1 beta-mediated hypoxic neuronal injury.

The purpose of this study was to elucidate the cellular/biochemical pathway(s) by which interleukin-1beta (IL-1beta) contributes to the pathogenesis of hypoxic-ischemic brain damage. In vivo, IL-1 receptor type I (IL-1RI)-deficient mice showed smaller infarcts and less neurological deficits than wild-type animals after a 90 min reversible middle cerebral artery occlusion. In vitro, IL-1beta mediated an enhancement of hypoxic neuronal injury in murine cortical cultures that was lacking in cultures derived from IL-1RI null mutant animals and was blocked by the IL-1 receptor antagonist or an IL-1RI blocking antibody. This IL-1beta-mediated potentiation of hypoxic neuronal injury was associated with an increase in both cellular cystine uptake ([cystine]i) and extracellular glutamate levels ([glutamate]e) and was prevented by either ionotropic glutamate receptor antagonism or removal of L-cystine, suggesting a role for the cystine/glutamate antiporter (System x(c)-). Indeed, dual System x(c)-/metabotropic glutamate receptor subunit 1 (mGluR1) antagonism but not selective mGluR1 antagonism prevented neuronal injury. Additionally, cultures derived from mGluR1-deficient mice exhibited the same potentiation in injury after treatment with IL-1beta as wild-type cultures, an effect prevented by System x(c)-/mGluR1 antagonism. Finally, assessment of System x(c)- function and kinetics in IL-1beta-treated cultures revealed an increase in velocity of cystine transport (Vmax), in the absence of a change in affinity (Km). Neither the enhancement in [cystine]i, [glutamate]e, or neuronal injury were observed in chimeric cultures consisting of IL-1RI(+/+) neurons plated on top of IL-1RI(-/-) astrocytes, highlighting the importance of astrocyte-mediated alterations in System x(c)- as a novel contributor to the development and progression of hypoxic neuronal injury.

Interleukin-1beta potentiates neuronal injury in a variety of injury models involving energy deprivation.

The purpose of this study was to develop a suitable in vitro model system to study the biochemical pathway(s) by which interleukin-1beta (IL-1beta) contributes to the pathogenesis of cerebral ischemia. Thus, the effect of IL-1beta on a number of injury paradigms associated with energy deprivation was investigated using murine mixed cortical cell cultures. While IL-1beta by itself was not neurotoxic, pre-treatment-but not concurrent or post-treatment-with this cytokine potentiated neuronal injury induced by depriving cultures of either oxygen, glucose, or both oxygen and glucose. Cytotoxicity was abolished by an IL-1beta-neutralizing antibody. Together, these results demonstrate the establishment of reliable and reproducible in vitro models that will now allow detailed investigation of the cellular and molecular mechanisms relating to IL-1beta-mediated neuronal cell death.