The use of recovery animals in nonclinical safety assessment studies with monoclonal antibodies: further 3Rs opportunities remain.

Assessment of reversibility from nonclinical toxicity findings in animals with potential adverse clinical impact is required during pharmaceutical development, but there is flexibility around how and when this is performed and if recovery animals are necessary. For monoclonal antibodies (mAbs) and in accordance with ICH S6(R1) if inclusion of recovery animals is warranted, this need only occur in one study. Data on study designs for first-in-human (FIH)-enabling and later-development toxicity studies were shared from a recent collaboration between the NC3Rs, EPAA, Netherlands Medicines Evaluation Board (MEB) and 14 pharmaceutical companies. This enabled a review of practices on recovery animal use during mAb development and identification of opportunities to reduce research animal use. Recovery animals were included in 68% of FIH-enabling and 69% of later-development studies, often in multiple studies in the same program. Recovery groups were commonly in control plus one test article-dosed group or in all dose groups (45% of studies, each design). Based on the shared data review and conclusions, limiting inclusion of recovery to a single nonclinical toxicology study and species, study design optimisation and use of existing knowledge instead of additional recovery groups provide opportunities to further reduce animal use within mAb development programs.

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.

Cross-company evaluation of the human lymphocyte activation assay.

Nonclinical immunotoxicity evaluation is an important component of safety assessment for pharmaceuticals. One in vitro assay that can be applied in a weight of evidence assessment is the human lymphocyte activation (HuLA) assay, an antigen recall assay, similar in many respects to the in vivo T-cell-dependent antibody response (TDAR) in that cooperation of multiple immune cell types are needed to produce responses. This assay uses human cells and is more amenable than the TDAR to compound ranking and mechanistic studies. The HuLA assay requires less time and drug than TDAR assays, uses a relevant antigen (influenza), reflects a human immune response, and applies principles of the 3Rs to non-clinical safety assessment. Peripheral blood mononuclear cells (PBMC) from flu-immunized donors are re-stimulated with flu-vaccine in the presence of test articles, and proliferation is measured. Published data demonstrate the applicability of the HuLA assay, but it has not been evaluated for reproducibility across testing sites. To evaluate assay reproducibility, scientists from a consortium of institutions conducted the assay in parallel, using a common pool of donor PBMC, influenza vaccine, and known immunosuppressant compounds (cyclosporine A and mycophenolic acid). The HuLA assay was highly reproducible in identification of inhibition of antigen-specific responses, and there was significant agreement across testing sites in the half maximal inhibitory concentration (IC50) values. Intra-site variability was the largest contributor to the variability observed within the assay. The HuLA assay was demonstrated to be ideally suited to comparing multiple compounds (i.e. compound ranking or benchmarking) within the same assay. Overall, the data reported herein support the HuLA assay as a useful tool in mechanistic evaluations of antigen-specific immune responses.

Methods: implementation of in vitro and ex vivo phagocytosis and respiratory burst function assessments in safety testing.

Functional innate immune assessments, including phagocytosis and respiratory burst, are at the forefront of immunotoxicology evaluation in pre-clinical animal species. Although in the clinic and in academic science, phagocytosis, and respiratory burst assessments have been reported for over two decades, the implementation of phagocytosis and respiratory burst analyses in toxicology safety programs is just recently gaining publicity. Discussed herein are general methods, both microtiter plate-based and flow cytometric-based, for assessing phagocytosis and respiratory burst in pre-clinical species including mouse, rat, dog, and monkey. This methods-centric discussion includes a review of technologies and descriptions of method applications, with examples of results from analyses testing reported inhibitors (rottlerin, wortmannin, and SB203580) of phagocytosis and respiratory burst. Justification of implementation, strategic experimental design planning, and feasibility aspects of evaluating test article effects on phagocytosis and respiratory burst function are described within the context of a case study. The case study involves investigation of the effects of a small molecule p38 kinase inhibitor, BMS-582949, on phagocytosis and respiratory burst functions in rat and monkey neutrophils and monocytes in vitro, as well as ex vivo in these innate immune cells from monkeys administered BMS-582949 during a 1-week repeat dose investigative study. The results of the in vitro and ex vivo assessments demonstrated that BMS-582949 inhibited phagocytosis and respiratory burst. These findings correlated with incidences of opportunistic infections observed in rat and monkey toxicity studies.

Viral host resistance studies.

A foremost objective of preclinical immunotoxicity testing is to address whether or not a drug or environmental toxicant causes adverse effects on net immune health, expressly the host's ability to mount an appropriate immune response to clear infectious organisms. Given the complex interactions, diverse molecular signaling events, and redundancies of immunity that has itself been subdivided into interdependent arms, namely innate, adaptive, and humoral, the results of single immune parameter testing may not reflect the final outcome of a drug or toxicant's effect on net immune health. The most comprehensive experimental approach to ascertain this information is utilization of host resistance models. Herein, application of viral host resistance models in rodents and non-human primates is described. Although brief descriptions of numerous viral models are discussed including reovirus, Epstein-Barr virus, cytomegalovirus, and lymphocryptovirus, the most well-characterized viral host resistance model, rodent influenza, is emphasized.

Selective leukemic-cell killing by a novel functional class of thalidomide analogs.

Using a novel cell-based assay to profile transcriptional pathway targeting, we have identified a new functional class of thalidomide analogs with distinct and selective antileukemic activity. These agents activate nuclear factor of activated T cells (NFAT) transcriptional pathways while simultaneously repressing nuclear factor-kappaB (NF-kappaB) via a rapid intracellular amplification of reactive oxygen species (ROS). The elevated ROS is associated with increased intracellular free calcium, rapid dissipation of the mitochondrial membrane potential, disrupted mitochondrial structure, and caspase-independent cell death. This cytotoxicity is highly selective for transformed lymphoid cells, is reversed by free radical scavengers, synergizes with the antileukemic activity of other redox-directed compounds, and preferentially targets cells in the S phase of the cell cycle. Live-cell imaging reveals a rapid drug-induced burst of ROS originating in the endoplasmic reticulum and associated mitochondria just prior to spreading throughout the cell. As members of a novel functional class of "redoxreactive" thalidomides, these compounds provide a new tool through which selective cellular properties of redox status and intracellular bioactivation can be leveraged by rational combinatorial therapeutic strategies and appropriate drug design to exploit cell-specific vulnerabilities for maximum drug efficacy.