Toxicology Testing of Biologics in Pre-Clinical Studies
Immunomodulatory biologics (“biologics”) are large molecules – proteins, peptides, and nucleotides – produced through biological processes in living cells and organisms. Currently, biologics represent one-third of newly licensed pharmaceutical products and target a broad range of diseases, mainly in oncology and autoimmune/inflammatory disorders. These biologics are generated through recombinant DNA-based techniques and are divided into 3 categories: monoclonal antibodies, replacement or modulators of signaling proteins, and replacement or modulators of cell surface receptors.
In the past two decades, the proportion of biologics-based approaches and drug approvals (relative to small molecules) have increased partially due to the biologics’ specificity to the intended target and predictable biodistribution, thus conferring a desirable safety advantage over small molecules. Nevertheless, several clinical concerns have emerged relating to unwanted effects associated with the use of biologics. In this blog, we will focus on toxicology testing for predicting safety and efficacy of biologics.
In the early development stage, researchers should generate pharmacokinetic and pharmacodynamics data and provide functional cross-reactivity of the biologic with at least one representative toxicology species. Non-human primate species are considered to be the most relevant in vivo model for attaining such data as they are the most similar to the human immune system.
An alternative approach is to develop a transgenic mouse model in which the human target protein (e.g., CD20 in arthritis models) is expressed and can be modulated by the candidate biologic. When species-specific biologics need to be assessed, surrogate or homologous molecules are often applied in pre-clinical testing for evaluating therapeutic efficacy as well as for predicting adverse reactions.
Biologic-induced adverse reactions are broadly categorized into two groups: 1. Adverse reactions associated with exaggerated immune activation, including serious infections, malignancies, and cytokine release syndrome; 2. Humoral and/or cellular immune responses (i.e., immunogenicity), which induce the production of neutralizing anti-drug antibodies, thus impeding therapeutic efficacy.
Owing to the complex interactions among disease states, the immune system, and biologics, the predictive tests to be used are determined on a case-by-case basis. We list here some of the current tools for predicting biologic-induced unwanted adverse reactions:
Predicting serious infections and malignancies
Within vivo experimental systems, biologic-induced immune perturbations can be evaluated by general immunological status – such as leukocyte counts and globulin levels – and immunophenotyping of lymphocytes from blood or lymphoid tissues (e.g., T, B, and NK cells) by using flow cytometry.
The T cell-dependent antibody response assay using a T cell-dependent antigen (e.g., keyhole limpet hemocyanin in rodents) can be applied to identify biologic-induced immunosuppression. These above assays provide information on the specific dysregulation occurring in the immune system and are useful tools for predicting human susceptibility to immunity-related infections and malignancies.
Predicting cytokine release syndrome
Cytokine release syndrome is characterized by the uncontrolled release of proinflammatory cytokines – such as interleukin 6 and IFNγ – mediated by the biologic. In vitro testing with human blood cells in the whole-blood assay or the peripheral blood mononuclear cell-based assay is one of the currently available approaches for predicting cytokine release syndrome.
In the preclinical stage, conventional repeat-dose toxicity testing can be carried out to examine whether there is immunogenicity that could significantly compromise exposure with longer term dosing. Other preclinical tools involve the use of in silico methods that identify potential immunogenic epitopes in biologics. Regarding in vivo strategies, the immune system-humanized mouse model and the human immune system xenograft mouse model have been developed for testing the immunogenic potential of biologics.
Developing suitable toxicology testing that recapitulate effects by a biologic in large patient populations remains a considerable challenge, suggesting a continued need to explore and validate predictive models. When tackling such a task in the design of appropriate toxicology models for a biologic, researchers should embrace knowledge of inter-species similarities and differences in all aspects of pharmacology and immunology of biologics so that obtained data can warn of potential risks and, more likely, be effectively translated to humans.
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