Translation of nonclinical to clinical safety findings for 27 biotherapeutics.

Human adverse drug reactions (ADRs), and in vivo nonclinical adverse and nonadverse findings, were identified in 27 biotherapeutic programs and placed into organ categories to determine translation. The sensitivity of detecting human ADRs was 30.8% with a positive predictive value (PPV) of 53.3% for nonclinical adverse findings; sensitivity increased to 67.3% and PPV fell to 35.0% when including nonadverse findings. Nonclinical findings were associated with a greater likelihood of a human ADR in that organ category, especially for adverse findings [positive likelihood ratio (LR+) >10 (lower 95% confidence interval [CI] of >5)]. The specificity and negative predictive value (NPV) were very high (>85%). A lack of nonclinical findings in an organ category was associated with a lower likelihood of a human ADR in that organ category. About 40-50% of human ADRs and nonclinical adverse findings, and about 30% of nonclinical nonadverse findings, were attributed to pharmacology. Slightly more than half of the human ADRs with a translating nonclinical finding had findings in animals that could be considered very similar. Overall, 38% of nonclinical findings translated to a human ADR at the organ category level. When nonclinical findings did not translate to humans, the cause was usually higher exposures or longer dosing in animals. All programs with human ADRs attributed to immunogenicity also had nonclinical adverse or nonadverse findings related to immunogenicity. Overall, nonclinical adverse and nonadverse findings were useful in predicting human ADRs, especially at an organ category level, and the majority of human ADRs were predicted by nonclinical toxicity studies.

Immune-Mediated Liver Effects Associated With Administration of a Human Anti-IL-21 Receptor Antibody (ATR-107) in Rats.

The toxicity of ATR-107, a human anti-interleukin-21 receptor (IL-21R) monoclonal antibody (mAb), was evaluated in CD-1 mice and cynomolgus monkeys after single-dose intravenous (IV) administration, and in Sprague-Dawley (SD) rats and cynomolgus monkeys after weekly IV and subcutaneous (SC) administration in 13-week toxicity studies that included recovery. Adverse liver necrosis, diffuse bridging fibrosis, and higher liver enzymes occurred in rats in the low-dose IV group (10 mg/kg), but not at 50 or 250 mg/kg IV, and not following SC administration despite overlapping systemic ATR-107 exposures. Similar findings were not seen in mice or cynomolgus monkeys. A series of investigative rat toxicity studies showed liver findings only occurred after administration of at least 3 weekly doses, only occurred in rats that developed anti-drug antibodies (ADAs), and the incidence was associated with higher ADAs titers. However, the presence of ADAs did not always result in liver injury. Liver findings did not occur in nude rats, which had high ATR-107 exposures and no ADAs. These findings suggest an adaptive immune response with formation of ADAs was necessary for development of ATR-107-related liver findings, and that liver injury can occur in rats secondary to development of ADAs following repeated administration of a human therapeutic mAb.

Opportunities and Challenges for Use of Minipigs in Nonclinical Pharmaceutical Development: Results of a Follow-Up IQ DruSafe Survey.

Minipigs are valid nonrodent species infrequently utilized for pharmaceutical research and development (R&D) compared with dogs or nonhuman primates (NHPs). A 2022 IQ DruSafe survey revealed a modest increase in minipig use by pharmaceutical companies compared with a prior 2014 survey, primarily in the development of oral small molecules and parenteral protein molecules. Some companies considered using minipigs more often due to NHP shortages and regional ethical concerns with using NHPs and dogs. However, for most pharmaceutical companies, minipigs still represent ≤5% of their nonrodent animal use. Key challenges noted by companies to wider adoption of minipigs were high test article requirement, limited historical control data, and lack of relevant reagents or assays. Additionally, some companies expressed uncertainties about contract research organization (CRO) capabilities and experience, a perception not shared by respondent CROs. These latest survey results indicate persistence of many concerns previously identified in 2014. Several case studies are included to illustrate areas of expanded minipig use as well as the challenges that hinder broader adoption. Ongoing, focused, and industry-wide initiatives to address the identified or perceived challenges may lead to more frequent or routine consideration of minipigs as a test species in pharmaceutical R&D.

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.

Re-evaluating the need for chronic toxicity studies with therapeutic monoclonal antibodies, using a weight of evidence approach.

To support registration of monoclonal antibodies (mAbs) for chronic indications, 6-month toxicity studies have historically been conducted. Experience with mAb development has shown a relatively benign and well-understood safety profile for this class, with most toxicity findings anticipated based on pharmacology. We evaluated whether a 6-month toxicity study is necessary to assess the long-term safety of mAbs. Data on First-in-Human (FIH)-enabling and chronic toxicity studies were shared for 142 mAbs submitted by 11 companies. Opportunities to further optimize study designs to reduce animal usage were identified. For 71% of mAbs, no toxicities or no new toxicities were noted in chronic studies compared to FIH-enabling study findings. New toxicities of potential concern for human safety or that changed trial design were identified in 13.5% of cases, with 7% being considered critical and 2% leading to program termination. An iterative, weight-of-evidence model which considers factors that influence the overall risk for a mAb to cause toxicity was developed. This model enables an evidence-based justification, suggesting when 3-month toxicity studies are likely sufficient to support late-stage clinical development and registration for some mAbs.

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.

Comparative nonclinical assessments of the biosimilar PF-06410293 and originator adalimumab.

Adalimumab, a recombinant fully human monoclonal antibody targeting tumor necrosis factor (TNF), is approved in the United States and Europe to treat various inflammatory and autoimmune indications. Biosimilars are approved biologics highly similar, but not identical, to approved biotherapeutics. To support clinical development of PF-06410293, an adalimumab biosimilar, nonclinical studies evaluated the structural, functional, toxicologic, and toxicokinetic similarity to originator adalimumab sourced from the United States (adalimumab-US) and European Union (adalimumab-EU). Structural similarity was assessed by peptide mapping. Biologic activity was measured via inhibition of TNF-induced apoptosis and Fc-based functionality assessments. In vivo nonclinical similarity was evaluated in a toxicity study in cynomolgus monkeys administered subcutaneous PF-06410293 or adalimumab-EU (0 or 157 mg/kg/week). Peptide mapping demonstrated PF-06410293, adalimumab-US, and adalimumab-EU had identical amino acid sequences. Comparative functional and binding assessments were similar. Effects of PF-06410293 and adalimumab-EU were similar and limited to pharmacologically mediated decreased cellularity of lymphoid follicles and germinal centers in spleen. Toxicokinetics were similar; maximum plasma concentration and area-under-the-concentration-time curve ratio of PF-06410293:adalimumab-EU ranged from 1.0 to 1.2. These studies supported PF-06410293 entry into clinical development. Many regulatory agencies now only request nonclinical in vivo testing if there is residual uncertainty regarding biosimilarity after in vitro analytical studies.

Nonclinical assessments of the potential biosimilar PF-06439535 and bevacizumab.

Bevacizumab, a recombinant humanized monoclonal antibody targeting vascular endothelial growth factor (VEGF), is approved for treatment of metastatic colorectal cancer, nonsquamous non-small-cell lung cancer, metastatic kidney cancer, and glioblastoma. To support clinical development of the potential bevacizumab biosimilar PF-06439535, nonclinical studies evaluated structural, functional, toxicological, and toxicokinetic similarity to bevacizumab sourced from the European Union (bevacizumab-EU) and United States (bevacizumab-US). Peptide mapping demonstrated the amino acid sequence of PF-06439535 was identical to bevacizumab-EU and bevacizumab-US. Biologic activity, measured via inhibition of VEGF-induced cell proliferation in human umbilical vein endothelial cells and binding to VEGF isoforms, was similar across the three drugs. In vivo similarity was demonstrated in cynomolgus monkeys administered intravenous PF-06439535 or bevacizumab-EU (0 or 10 mg/kg/dose twice weekly for 1 month; total of nine doses). Systemic exposure appeared similar and test article-related effects were limited to physeal dysplasia of the distal femur. The potential for non-target-mediated toxicity of PF-06439535 was evaluated in rats administered intravenous PF-06439535 (15 or 150 mg/kg/dose twice weekly for 15 days; total of five doses). Nonadverse higher liver weights and minimal sinusoidal cell hyperplasia were observed. Collectively, these studies demonstrated similarity of PF-06439535 to bevacizumab, supporting entry into clinical development.

A Synopsis of the "Influence of Epigenetics, Genetics, and Immunology" Session Part A at the 35th Annual Society of Toxicologic Pathology Symposium.

The overarching theme of the 2016 Society of Toxicology Pathology's Annual Symposium was "The Basis and Relevance of Variation in Toxicologic Responses." Session 4 focused on genetic variation as a potential source for variability in toxicologic responses within nonclinical toxicity studies and further explored how knowledge of genetic traits might enable targeted prospective and retrospective studies in drug development and human health risk assessment. In this session, the influence of both genetic sequence variation and epigenetic modifications on toxicologic responses and their implications for understanding risk were explored. In this overview, the presentations in this session will be summarized, with a goal of exploring the ramifications of genetic and epigenetic variability within and across species for toxicity studies and disseminating information regarding novel tools to harness this variability to advance understanding of toxicologic responses across populations.

Nonproliferative and Proliferative Lesions of the Gastrointestinal Tract, Pancreas and Salivary Glands of the Rat and Mouse.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) project is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature and diagnostic criteria for nonproliferative and proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature and diagnostic criteria for classifying lesions in the digestive system including the salivary glands and the exocrine pancreas of laboratory rats and mice. Most lesions are illustrated by color photomicrographs. The standardized nomenclature, the diagnostic criteria, and the photomicrographs are also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and age related lesions as well as lesions induced by exposure to test items. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature and diagnostic criteria for the digestive system will decrease misunderstandings among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

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.

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.

Immunogenicity/hypersensitivity of biologics.

This continuing education course was designed to provide an overview of the immunologic mechanisms involved in immunogenicity and hypersensitivity reactions following administration of biologics in nonclinical toxicity studies, the methods used to determine whether such reactions are occurring, and the associated clinical and anatomic pathology findings. Hypersensitivity reactions have classically been divided into type I, II, III, and IV reactions; type I and III reactions are those most often observed following administration of biologics. A variety of methods can be used to detect these reactions. Antemortem methods include hematology; detection of antidrug antibodies, circulating immune complexes and complement fragments, and immunoglobulin E in serum; tests for serum complement activity; and evaluation of complement receptor 1 on erythrocytes. Postmortem methods include routine light microscopy and electron microscopy, which can demonstrate typical findings associated with hypersensitivity reactions, and immunohistochemistry, which can detect the presence of immune complexes in tissues, including the detection of the test article. A final determination of whether findings are related to a hypersensitivity reaction in individual animals or across the entire study should rely on the overall weight of evidence, as findings indicative of these reactions are not necessarily consistent across all affected animals.

Comparative nonclinical assessments of the proposed biosimilar PF-05280586 and rituximab (MabThera®).

Comparative nonclinical studies were conducted with the proposed biosimilar PF-05280586 and rituximab-EU (MabThera®). In side-by-side analyses, peptide maps and complement-dependent cytotoxicity assay results were similar. Sexually-mature cynomolgus monkeys were administered PF-05280586 or rituximab-EU as a single dose of 0, 2, 10, or 20 mg/kg on day 1 and observed for 92 days (single-dose study) or as 5 weekly injections of 0 or 20 mg/kg and necropsied on day 30, the day after the 5th dose, or on day 121 (repeat-dose study). The pharmacokinetic and pharmacodynamic profiles for both molecules were similar. Marked depletion of peripheral blood B cells 4 days after dosing was followed by near or complete repletion (single-dose study) or partial repletion (repeat-dose study). In the single-dose study, anti-drug antibodies (ADA) were detected by day 29 in all animals administered PF-05280586 or rituximab-EU and persisted through day 85, the last day tested. In the repeat-dose study, ADA were detected on day 121 in 50% of animals administered PF-05280586 or rituximab-EU. Both molecules were well tolerated at all doses. In all endpoints evaluated, PF-05280586 exhibited similarity to rituximab-EU.

"Candidatus Mycoplasma haemomacaque" and Bartonella quintana bacteremia in cynomolgus monkeys.

Here, we report latent infections with Bartonella quintana and a hemotropic Mycoplasma sp. in a research colony of cynomolgus monkeys (Macaca fascicularis). Sequence alignments, evolutionary analysis, and signature nucleotide sequence motifs of the hemotropic Mycoplasma 16S rRNA and RNase P genes indicate the presence of a novel organism.

Regulatory forum opinion piece: differences between protein-based biologic products (biotherapeutics) and chemical entities (small molecules) of relevance to the toxicologic pathologist.

With the advances in cell culture methodologies and molecular biology that have occurred over the past several decades, biologics have become as common as small molecules within the portfolios of the pharmaceutical industry. Toxicologic pathologists should be aware of some of the fundamental differences between small molecules and biologics. Effects are not always observed in studies following administration of biologics. When findings are observed, the toxicologic pathologist should initially determine whether the effect(s) are mediated (directly or indirectly) via the intended pharmacology, exaggerated pharmacology, an immune response, and/or off target effects. Following this determination, the toxicologic pathologist should provide an assessment regarding the relevance of the findings to the intended clinical population, usually humans. The toxicologic pathologist may also be asked to assess unusual species and models. Given their broad background in physiology and immunology, toxicologic pathologists are uniquely positioned to provide this input to drug development teams.

Cytosolic phospholipase A2α blockade abrogates disease during the tissue-damage effector phase of experimental autoimmune encephalomyelitis by its action on APCs.

Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to PGs via the cyclooxygenase 1 and 2 pathways and to leukotrienes via the 5-lipoxygenase pathway. We used adoptive transfer and relapsing-remitting forms of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, in two different strains of mice (SJL or C57BL/6) to demonstrate that blockade of cPLA(2)α with a highly specific small-molecule inhibitor during the tissue-damage effector phase abrogates the clinical manifestation of disease. Using the adoptive transfer model in SJL mice, we demonstrated that the blockade of cPLA(2)α during the effector phase of disease was more efficacious in ameliorating the disease pathogenesis than the blockade of each of the downstream enzymes, cyclooxygenase-1/2 and 5-lipooxygenase. Similarly, blockade of cPLA(2)α was highly efficacious in ameliorating disease pathogenesis during the effector phase of EAE in the adoptive transfer model of EAE in C57BL/6 mice. Investigation of the mechanism of action indicates that cPLA(2)α inhibitors act on APCs to diminish their ability to induce Ag-specific effector T cell proliferation and proinflammatory cytokine production. Furthermore, cPLA(2)α inhibitors may prevent activation of CNS-resident microglia and may increase oligodendrocyte survival. Finally, in a relapsing-remitting model of EAE in SJL mice, therapeutic administration of a cPLA(2)α inhibitor, starting from the peak of disease or during remission, completely protected the mice from subsequent relapses.

Reproductive performance and early postnatal development in interleukin (IL)-13-deficient mice.

OBJECTIVE: The purpose of this study was to assess the effect of interleukin (IL)-13 deficiency on fertility and reproductive performance of adult mice and on morphological and behavioral development of the offspring. METHODS: Wild-type and homozygous IL-13-deficient (KO) mice were grouped by genotype, and male and female mice were mated within each group. Adult (F(0) ) mice were evaluated for reproductive performance, and development was assessed in F(1) fetuses on gestation day 18, and in F(1) pups to postnatal day 35. RESULTS: In F(0) males, there were no differences in the number of males that mated or impregnated females, or in total sperm count or sperm motility, between the wild-type and KO groups. In F(0) females, there were no observed genotype-related differences in fertility, length of gestation, number of viable fetuses per litter, or viability of offspring. There were no differences in embryo-fetal development (external/palate, skeletal, visceral) of the F(1) fetuses between genotypes. Similarly, IL-13 deficiency had no impact on any postnatal parameters assessed including reflex, sexual maturation, learning, and memory. CONCLUSIONS: IL-13 deficiency had no observed effect on reproductive performance or morphological and behavioral development in mice.

Cytosolic phospholipase A2 alpha-deficient mice are resistant to experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the central nervous system (CNS), is a model of human multiple sclerosis. Cytosolic phospholipase A2alpha (cPLA2alpha), which initiates production of prostaglandins, leukotrienes, and platelet-activating factor, is present in EAE lesions. Using myelin oligodendrocyte glycoprotein (MOG) immunization, as well as an adoptive transfer model, we showed that cPLA2alpha-/- mice are resistant to EAE. Histologic examination of the CNS from MOG-immunized mice revealed extensive inflammatory lesions in the cPLA2alpha+/- mice, whereas the lesions in cPLA2alpha-/- mice were reduced greatly or completely absent. MOG-specific T cells generated from WT mice induced less severe EAE in cPLA2alpha-/- mice compared with cPLA2alpha+/- mice, which indicates that cPLA2alpha plays a role in the effector phase of EAE. Additionally, MOG-specific T cells from cPLA2alpha-/- mice, transferred into WT mice, induced EAE with delayed onset and lower severity compared with EAE that was induced by control cells; this indicates that cPLA2alpha also plays a role in the induction phase of EAE. MOG-specific T cells from cPLA2alpha-/- mice were deficient in production of Th1-type cytokines. Consistent with this deficiency, in vivo administration of IL-12 rendered cPLA2alpha-/- mice susceptible to EAE. Our data indicate that cPLA2alpha plays an important role in EAE development and facilitates differentiation of T cells toward the Th1 phenotype.

Survey results on the use of the tissue cross-reactivity immunohistochemistry assay.

A multinational pharmaceutical and biotechnology company survey was conducted to gain a better understanding of the use and value of the tissue cross-reactivity (TCR) assay in the development of biotherapeutic molecules. The majority of the molecules did not use TCR data as the only basis for determining species selection for toxicity studies (73%). For 95% of the molecules, the TCR data had no impact on the development strategy. For 2% of the molecules (1/56), TCR data was the sole source of information indicating a potential risk to patients. Unexpected or off-target binding was seen with 35% of the molecules, with the majority of this binding occurring in the CNS and reproductive organs. Tissues that were known or presumed to contain the target stained positively in 22% and 10% of molecules tested in non-human primate and human tissues, respectively. Tissues that were known or presumed to lack the target were negative for staining in 39% and 50% of molecules for non-human primate and human tissue, respectively. For 5% (6/110) of all the molecules, companies stated that toxicities would have been missed in animal studies or the clinic (i.e., not identified by clinical signs, histopathology, etc.) if the TCR studies had not been performed.