Challenges of non-clinical safety testing for biologics: A Report of the 9th BioSafe European Annual General Membership Meeting.

New challenges and other topics in non-clinical safety testing of biotherapeutics were presented and discussed at the nineth European BioSafe Annual General Membership meeting in November 2019. The session topics were selected by European BioSafe organization committee members based on recent company achievements, agency interactions and new data obtained in the non-clinical safety testing of biotherapeutics, for which data sharing would be of interest and considered as valuable information. The presented session topics ranged from strategies of in vitro testing, immunogenicity prediction, bioimaging, and developmental and reproductive toxicology (DART) assessments to first-in-human (FIH) dose prediction and bioanalytical challenges, reflecting the entire space of different areas of expertise and different molecular modalities. During the 9th meeting of the European BioSafe members, the following topics were presented and discussed in 6 main sessions (with 3 or 4 presentations per session) and in three small group breakout sessions: 1) DART assessment with biotherapeutics: what did we learn and where to go?; 2) Non-animal testing strategies; 3) Seeing is believing: new frontiers in imaging; 4) Predicting immunogenicity during early drug development: hope or despair?; 5) Challenges in FIH dose projections; and 6) Non-canonical biologics formats: challenges in bioanalytics, PKPD and biotransformation for complex biologics formats. Small group breakout sessions were organized for team discussion about 3 specific topics: 1) Testing of cellular immune function in vitro and in vivo; 2) MABEL approach (toxicology and pharmacokinetic perspective); and 3) mRNA treatments. This workshop report presents the sessions and discussions at the meeting.

Nonclinical Development of Biologics: Integrating Safety, Pharmacokinetics, and Pharmacodynamics to Create Smarter and More Flexible Nonclinical Safety Programs Optimizing Animal Use.

Safety assessment of biological drugs has its challenges due to the multiple new different modalities, for example, antibody-drug conjugates, bispecifics, nanobodies, fusion proteins and advanced therapy medicinal products (ATMPs), their different pharmacokinetic and pharmacodynamic properties, and their ability to trigger immunogenicity and toxicity. In the public and in the pharmaceutical industry, there is a strong and general desire to reduce the number of animals used in research and development of drugs and in particular reducing the use of nonhuman primates. Important discussions and activities are ongoing investigating the smarter designs of early research and dose range finding studies, reuse of animals, and replacing animal experiments with in vitro studies. Other important challenges include absence of a relevant species and design of studies and developing genetically modified animals for special investigative toxicology studies. Then, the learnings and challenges from the development of the first ATMPs are available providing valuable insights in the development path for these new potentially transformative treatments. Finally, development of strategies for assessment of immunogenicity and prediction of translation of immunogenicity and associated findings to the clinic. On this, the eighth meeting for the European BioSafe members, these challenges served as the basis for the presentations and discussions during the meeting. This article serves as the workshop report reviewing the presentations and discussions at the meeting.

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.

Biotherapeutics in non-clinical development: Strengthening the interface between safety, pharmacokinetics-pharmacodynamics and manufacturing.

Biological drugs comprise a wide field of different modalities with respect to structure, pharmacokinetics and pharmacological function. Considerable non-clinical experience in the development of proteins (e.g. insulin) and antibodies has been accumulated over the past thirty years. In order to improve the efficacy and the safety of these biotherapeutics, Fc modifications (e.g. Fc silent antibody versions), combinations (antibody-drug conjugates, protein-nanoparticle combinations), and new constructs (darpins, fynomers) have been introduced. In the last decade, advanced therapy medicinal products (ATMPs) in research and development have become a considerable and strongly growing part of the biotherapeutic portfolio. ATMPs consisting of gene and cell therapy modalities or even combinations of them, further expand the level of complexity, which already exists in non-clinical development strategies for biological drugs and has thereby led to a further diversification of expertise in safety and PKPD assessment of biological drugs. It is the fundamental rationale of the BioSafe meetings, held yearly in the EU and in the US, to convene experts on a regular basis and foster knowledge exchange and mutual understanding in this fast growing area. In order to reflect at least partially the variety of the biotherapeutics field, the 2016 EU BioSafe meeting addressed the following topics in six sessions: (i) In vitro Meets in vivo to Leverage Biologics Development (ii) New developments and regulatory considerations in the cell and gene therapy field (iii) CMC Challenges with Biologics development (iv) Minipigs in non-clinical safety assessment (v) Opportunities of PKPD Assessment in Less Common Administration Routes In the breakout sessions the following questions were discussed: (i) Cynomolgus monkey as a reprotoxicology Species: Impact of Immunomodulators on Early Pregnancy Maintenance (ii) Safety Risk of Inflammation and Autoimmunity Induced by Immunomodulators (iii) Experience with non-GMP Material in Pivotal Non-clinical Safety Studies to Support First in Man (FiM) Trials (iv) Safety Assessment of Combination Products for Non-oncology.

In vitro assays supporting the safety assessment of immunomodulatory monoclonal antibodies.

Many monoclonal antibodies (mAbs) licensed for human use or in clinical development for cancer and autoimmune disease directly interact with the immune system. These immunomodulatory mAbs have an inherent risk of adverse immune-mediated drug reactions, including infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the potential for immunotoxicity of a mAb is required to support administration to humans. This review will highlight the key role of in vitro assays in defining the immunopharmacology, immunotoxicity and immunogenicity of mAbs. A wide range of in vitro tests with multiple formats of different complexity can be utilized to characterize i) the antibody-binding domains of the mAb, such as on-target binding and downstream pharmacological effects (e.g. immunosuppression, immune activation, cytokine release) in both humans and animal species used for toxicology studies and off-target binding; ii) Fc-dependent effects such as Fc-mediated cellular activation (e.g. of leukocytes, platelets) and cytokine release, complement activation; and iii) product-related factors (sequence, physical-chemical properties and impurities) that can impact both pharmacological activity and immunogenicity potential of a mAb. These assays can be crucial to the selection of mAbs with an optimum balance of safety and efficacy, in defining whether a mAb is a high risk molecule, and together with animal data, can inform human safe starting doses and escalation schemes.

Translational immunotoxicology of immunomodulatory monoclonal antibodies.

While immunomodulatory monoclonal antibodies (mAbs) have a wide therapeutic potential, exaggerated immunopharmacology may drive both acute and delayed immunotoxicity. The existing tools for immunotoxicity assessment do not accurately predict the full range of immunotoxicities observed in humans. New and optimized models, assays, endpoints and biomarkers in animals and humans are required to safeguard patients and allow them access to these often transformational therapies.

Nonclinical safety testing of biopharmaceuticals--Addressing current challenges of these novel and emerging therapies.

Non-clinical safety testing of biopharmaceuticals can present significant challenges to human risk assessment with these often innovative and complex drugs. Hot Topics in this field were discussed recently at the 4th Annual European Biosafe General Membership meeting. In this feature article, the presentations and subsequent discussions from the main sessions are summarized. The topics covered include: (i) wanted versus unwanted immune activation, (ii) bi-specific protein scaffolds, (iii) use of Pharmacokinetic (PK)/Pharmacodynamic (PD) data to impact/optimize toxicology study design, (iv) cytokine release and challenges to human translation (v) safety testing of cell and gene therapies including chimeric antigen receptor T (CAR-T) cells and retroviral vectors and (vi) biopharmaceutical development strategies encompassing a range of diverse topics including optimizing entry of monoclonal antibodies (mAbs) into the brain, safety testing of therapeutic vaccines, non-clinical testing of biosimilars, infection in toxicology studies with immunomodulators and challenges to human risk assessment, maternal and infant anti-drug antibody (ADA) development and impact in non-human primate (NHP) developmental toxicity studies, and a summary of an NC3Rs workshop on the future vision for non-clinical safety assessment of biopharmaceuticals.

Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies.

Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease and as such, are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human starting dose is discussed.