Methodologies and Emerging Technologies for the Evaluation of the Hematopoietic System.

Hematology and bone marrow analysis is central to our understanding of the hematopoietic system and how it responds to insults, and this session presented during the 2022 STP symposium provided a review of current and novel approaches for the evaluation of the hematopoietic system in the context of nonclinical investigations. This publication summarizes the information presented on novel approaches for evaluation of the hematopoietic system using automated hematology analyzers, including details around the quantitative assessment of bone marrow cell suspensions as well as introducing several newly available hematology parameters. It was followed by a discussion on intravital microscopy and live cell imaging and how these methods can assist with de-risking hematopoiesis-associated safety concerns, and a review of recent assays using artificial intelligence for the evaluation of bone marrow.

Development of a nonhuman primate challenge model to evaluate CD8+ T cell responses to an adenovirus-based vaccine expressing SIV proteins upon repeat-dose treatment with checkpoint inhibitors.

Targeting immune checkpoint receptors expressed in the T cell synapse induces active and long-lasting antitumor immunity in preclinical tumor models and oncology patients. However, traditional nonhuman primate (NHP) studies in healthy animals have thus far demonstrated little to no pharmacological activity or toxicity for checkpoint inhibitors (CPIs), likely due to a quiescent immune system. We developed a NHP vaccine challenge model in Mauritius cynomolgus monkey (MCMs) that elicits a strong CD8+ T cell response to assess both pharmacology and safety within the same animal. MHC I-genotyped MCMs were immunized with three replication incompetent adenovirus serotype 5 (Adv5) encoding Gag, Nef and Pol simian immunodeficiency virus (SIV) proteins administered 4 weeks apart. Immunized animals received the anti-PD-L1 atezolizumab or an immune checkpoint-targeting bispecific antibody (mAbX) in early development. After a single immunization, Adv5-SIVs induced T-cell activation as assessed by the expression of several co-stimulatory and co-inhibitory molecules, proliferation, and antigen-specific T-cell response as measured by a Nef-dependent interferon-γ ELIspot and tetramer analysis. Administration of atezolizumab increased the number of Ki67+ CD8+ T cells, CD8+ T cells co-expressing TIM3 and LAG3 and the number of CD4+ T cells co-expressing 4-1BB, BTLA, and TIM3 two weeks after vaccination. Both atezolizumab and mAbX extended the cytolytic activity of the SIV antigen-specific CD8+ T cell up to 8 weeks. Taken together, this vaccine challenge model allowed the combined study of pharmacology and safety parameters for a new immunomodulatory protein-based therapeutic targeting CD8+ T cells in an NHP model.

An Investigative Study of Pancreatic Exocrine Biomarkers, Histology, and Histomorphometry in Male Zucker Diabetic Fatty (ZDF) Rats Given Dulaglutide by Subcutaneous Injection Twice Weekly for 13 Weeks.

Glucagon-like peptide-1 (GLP-1) receptor agonist therapy has been implicated as a possible risk factor for acute pancreatitis in patients with type 2 diabetes. Dulaglutide is a long-acting GLP-1 receptor agonist in development for treatment of type 2 diabetes. The effects of dulaglutide were evaluated in male Zucker diabetic fatty (ZDF) rats to examine whether dulaglutide may induce or modulate pancreatitis. Rats were randomized to dose groups receiving twice-weekly subcutaneously administered dulaglutide 0.5, 1.5, and 5.0 mg/kg/dose (corresponding human plasma exposures following twice-weekly dosing are 3-, 8-, and 30-fold, respectively) for 13 weeks or to vehicle control. Following termination, serially trimmed sections of pancreases were stained with hematoxylin and eosin or co-stained with an epithelial marker and a marker of either proliferation or apoptosis. Efficacious reductions in glucose and hemoglobin A1c occurred at all dulaglutide doses. Lipase activity was unaffected, and there were modest increases in total and pancreatic amylase activities at all doses without individual microscopic inflammatory correlates. Microscopic dulaglutide-related pancreatic changes included increased interlobular ductal epithelium without ductal cell proliferation (≥0.5 mg/kg), increased acinar atrophy with/without inflammation (≥1.5 mg/kg), and increased incidence/severity of neutrophilic acinar pancreatic inflammation (5.0 mg/kg). In summary, dulaglutide treatment was associated with mild alterations in ductal epithelium and modest exacerbation of spontaneous lesions of the exocrine pancreas typically found in the ZDF rat model.

Best practices for evaluation of bone marrow in nonclinical toxicity studies.

This manuscript is intended to provide a best practice approach to accurately and consistently assess toxicant-induced bone marrow effects of test articles. In nonclinical toxicity studies, complete blood count data in conjunction with the histological examination of the bone marrow are recommended as the foundation for assessing the effect of test articles on the hematopoietic system. This approach alone can be used successfully in many studies. However, in some situations it may be necessary to further characterize effects on the different hematopoietic lineages, either by cytological or flow cytometric evaluation of the bone marrow. Both modalities can be used successfully, and which one is selected will depend on the expertise, preference of the facility, and the nature of the change in the bone marrow. Other specialized techniques such as clonogenic assays or electron microscopy are used rarely to further characterize hematotoxicity. The indications and techniques to successfully employ histological, cytological, or flow cytometric evaluation as well as clonogenic assays and electron microscopy are reviewed.

Best practices for evaluation of bone marrow in nonclinical toxicity studies.

This manuscript is intended to provide a best practice approach to accurately and consistently assess toxicant-induced bone marrow effects of test articles. In nonclinical toxicity studies, complete blood count data in conjunction with the histological examination of the bone marrow are recommended as the foundation for assessing the effect of test articles on the hematopoietic system. This approach alone can be used successfully in many studies. However, in some situations it may be necessary to further characterize effects on the different hematopoietic lineages, either by cytological or flow cytometric evaluation of the bone marrow. Both modalities can be used successfully, and which one is selected will depend on the expertise, preference of the facility, and the nature of the change in the bone marrow. Other specialized techniques such as clonogenic assays or electron microscopy are used rarely to further characterize hematotoxicity. The indications and techniques to successfully employ histological, cytological, or flow cytometric evaluation as well as clonogenic assays and electron microscopy are reviewed.