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.

Classic and evolving approaches to evaluating cross reactivity of mAb and mAb-like molecules - A survey of industry 2008-2019.

Monoclonal antibodies (mAbs) and mAb derivatives have become mainstay pharmaceutical modalites. A critical assessment is to ascertain the specificity of these molecules prior to human clinical trials. The primary technique for determining specificity has been the immunohistochemistry (IHC)-based "Tissue Cross-Reactivity" (TCR) assay, where the candidate molecule is applied to > 30 tissues to look for unexpected staining. In the last few years, however, non-IHC array-based platforms have emerged that allow for screening 75-80% of the human membrane proteome, indicating a viable alternative and/or addition to the IHC methods. The preclinical sciences subcommittee of the Biotechnology Innovation Organization (BIO), "BioSafe", conducted a survey of 26 BIO member companies to understand current sponsor experience with the IHC and array techniques. In the last ten years, respondents noted they have conducted more than 650 IHC TCR assays, largely on full length mAbs, with varying impacts on programs. Protein/cell arrays have been utilized by almost half of the companies and sponsors are gaining familiarity and comfort with the platform. Initial experience with recent versions of these arrays has been largely positive. While most sponsors are not prepared to eliminate the IHC TCR assay, growing experience with these alternatives allows them to confidently choose other approaches with or without TCR assays.

Nonclinical Evaluation of PF-06438179: A Potential Biosimilar to Remicade® (Infliximab).

INTRODUCTION: PF-06438179, a potential biosimilar to Remicade® (infliximab, Janssen Biotech, Inc.), is a chimeric mouse-human monoclonal antibody targeting human tumor necrosis factor alpha (TNF). METHODS: Analytical (small subset reported here) and nonclinical studies compared the structural, functional, and in vivo nonclinical similarity of PF-06438179 with Remicade sourced from the United States (infliximab-US) and/or European Union (infliximab-EU). RESULTS: The peptide map profiles were superimposable, and peptide masses were the same, indicating identical amino acid sequences. Data on post-translational modifications, biochemical properties, and biological function provided strong support for analytical similarity. Administration of a single intravenous (IV) dose (10 or 50 mg/kg) of PF-06438179 or infliximab-EU to male rats was well tolerated. There were no test article-related clinical signs or effects on body weight or food consumption. Systemic exposures [maximum drug concentration (C max) and area under the concentration-time curve (AUC)] in rats administered PF-06438179 or infliximab-EU were similar, with mean exposure ratio of PF-06438179 relative to infliximab-EU ranging from 0.88 to 1.16. No rats developed anti-drug antibodies. A 2-week IV toxicity study was conducted with once-weekly administration of 10 or 50 mg/kg of PF-06438179 to male and female rats. PF-06438179-related hyperplasia of sinusoidal cells occurred in the liver in rats administered 50 mg/kg, but was not adverse based on its minimal to mild severity. The no-observed adverse-effect level for PF-06438179 was 50 mg/kg. At this dose, C max was 1360 µg/mL and AUC at 168 h was 115,000 µg h/mL on day 8. CONCLUSIONS: The analytical and nonclinical studies have supported advancement of PF-06438179 into global comparative clinical trials. FUNDING: Pfizer Inc.

Waiving in vivo studies for monoclonal antibody biosimilar development: National and global challenges.

Biosimilars are biological medicinal products that contain a version of the active substance of an already authorised original biological medicinal product (the innovator or reference product). The first approved biosimilar medicines were small proteins, and more recently biosimilar versions of innovator monoclonal antibody (mAb) drugs have entered development as patents on these more complex proteins expire. In September 2013, the first biosimilar mAb, infliximab, was authorised in Europe. In March 2015, the first biosimilar (Zarxio™, filgrastim-sndz, Sandoz) was approved by the US Food and Drug Administration; however, to date no mAb biosimilars have been approved in the US. There are currently major differences between how biosimilars are regulated in different parts of the world, leading to substantial variability in the amount of in vivo nonclinical toxicity testing required to support clinical development and marketing of biosimilars. There are approximately 30 national and international guidelines on biosimilar development and this number is growing. The European Union's guidance describes an approach that enables biosimilars to enter clinical trials based on robust in vitro data alone; in contrast, the World Health Organization's guidance is interpreted globally to mean in vivo toxicity studies are mandatory. We reviewed our own experience working in the global regulatory environment, surveyed current practice, determined drivers for nonclinical in vivo studies with biosimilar mAbs and shared data on practice and study design for 25 marketed and as yet unmarketed biosimilar mAbs that have been in development in the past 5y. These data showed a variety of nonclinical in vivo approaches, and also demonstrated the practical challenges faced in obtaining regulatory approval for clinical trials based on in vitro data alone. The majority of reasons for carrying out nonclinical in vivo studies were not based on scientific rationale, and therefore the authors have made recommendations for a data-driven approach to the toxicological assessment of mAb biosimilars that minimises unnecessary use of animals and can be used across all regions of the world.

Scientific and Regulatory Policy Committee Points-to-consider Paper*: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part I. Biotherapeutics.

Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. Although DIVI in laboratory animal species has been well characterized for vasoactive small molecules, there is little available information regarding DIVI associated with biotherapeutics such as peptides/proteins or antibodies. Because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans and the lack of robust biomarkers of DIVI, preclinical DIVI findings can cause considerable delays in or even halt development of promising new drugs. This review discusses standard terminology, characteristics, and mechanisms of DIVI associated with biotherapeutics. Guidance and points to consider for the toxicologist and pathologist facing preclinical cases of biotherapeutic-related DIVI are outlined, and examples of regulatory feedback for each of the mechanistic types of DIVI are included to provide insight into risk assessment.

Scientific and Regulatory Policy Committee Points-to-consider Paper*: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part 2. Antisense Oligonucleotides.

Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. In recent years, DIVI has been occasionally observed in nonhuman primates given RNA-targeting therapeutics such as antisense oligonucleotide therapies (ASOs) during chronic toxicity studies. While DIVI in laboratory animal species has been well characterized for vasoactive small molecules, and immune-mediated responses against large molecule biotherapeutics have been well described, there is little published information regarding DIVI induced by ASOs to date. Preclinical DIVI findings in monkeys have caused considerable delays in development of promising new ASO therapies, because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans, and the lack of robust biomarkers of DIVI. This review of DIVI discusses clinical and microscopic features of vasculitis in monkeys, their pathogenic mechanisms, and points to consider for the toxicologist and pathologist when confronted with ASO-related DIVI. Relevant examples of regulatory feedback are included to provide insight into risk assessment of ASO therapies.

Effects of an antagonist of neurokinin receptors 1, 2 and 3 on reproductive hormones in male beagle dogs.

BACKGROUND: SCH 206272, a neurokinin 1, 2, and 3 receptor antagonist, administered to beagle dogs results in testicular toxicity. Therefore, a series of experiments were conducted to determine whether this observed toxicity was associated with changes in reproductive hormones and hypothalamic gonadotrophin releasing hormone (GnRH) levels. METHODS: Male beagle dogs were administered 30 mg/kg SCH 206272 for up to 7 days. Blood samples were collected at the end of the dosing period for reproductive hormone analysis. Male reproductive organs were stained with hematoxylin and eosin and the hypothalamus was stained for GnRH. RESULTS: Intact male dogs exhibited SCH 206272-related decreases in pulsatility and magnitude of luteinizing hormone (LH) and testosterone, which were associated with seminiferous tubule degeneration, oligospermia, and epithelial atrophy in the prostate gland. Neutered dogs also exhibited SCH 206272-related decreases in LH and FSH. In a subsequent reversibility study, intact male dogs exhibited decreased LH, testosterone, and FSH, which exhibited recovery by 2 weeks post-dosing; however, seminiferous tubule degeneration and oligospermia did not exhibit recovery by 2 weeks post-dosing. Dogs administered SCH 206272 also exhibited an increase in mean number of GnRH-containing neurons in the hypothalamus and an increase in GnRH mRNA/neuron, which exhibited recovery by 2 weeks post-dosing. CONCLUSIONS: SCH 206272-dosed dogs exhibited rapid decreases in reproductive hormones and subsequent testicular pathology. Collectively, these changes in hormone levels suggest that the observed SCH 206272-related reproductive tract findings are the result of alterations in hypothalamic-pituitary-gonadal function. However, a direct effect on the testes cannot be definitively ruled out.

Use of tissue cross-reactivity studies in the development of antibody-based biopharmaceuticals: history, experience, methodology, and future directions.

Tissue cross-reactivity (TCR) studies are screening assays recommended for antibody and antibody-like molecules that contain a complementarity-determining region (CDR), primarily to identify off-target binding and, secondarily, to identify sites of on-target binding that were not previously identified. At the present time, TCR studies involve the ex vivo immunohistochemical (IHC) staining of a panel of frozen tissues from humans and animals, are conducted prior to dosing humans, and results are filed with the initial IND/CTA to support first-in-human clinical trials. In some cases, a robust TCR assay cannot be developed, and in these cases the lack of a TCR assay should not prevent a program from moving forward. The TCR assay by itself has variable correlation with toxicity or efficacy. Therefore, any findings of interest should be further evaluated and interpreted in the context of the overall pharmacology and safety assessment data package. TCR studies are generally not recommended for surrogate molecules or for comparability assessments in the context of manufacturing/cell line changes. Overall, the design, implementation, and interpretation of TCR studies should follow a case-by-case approach.