Formation and Glomerular Deposition of Immune Complexes in Mice Administered Human Antibodies: Evaluation of Dose, Frequency, and Biomarkers.

Administration of human protein-based drugs to animals often leads to formation of antidrug antibodies (ADAs) that may form circulating immune complexes (CICs) with the dosed protein. Circulating immune complexes can activate and bind complement (cCICs), and if large amount of CICs or cCICs is formed, the clearance mechanism potentially becomes saturated, which can lead to immune complex (IC) deposition and inflammation. To obtain a better understanding of the underlying factors, including the relationship between different dose regimes on IC formation and deposition and identification of possible biomarkers of IC deposition and IC-related pathological changes in kidneys, BALB/c and C57BL/6J mice were administered with human anti-tumor necrosis factor α (aTNFα, adalimumab) or a humanized anti-TNP (aTNP) antibody for 13 weeks. Particularly, ADA, CIC, cCIC formation, IC deposition, and glomerulonephritis were observed in C57BL/6J administered with aTNFα, whereas the immunologic response was minor in BALB/c mice administered with aTNFα and in BALB/c and C57BL/6J mice administered aTNP. Changing dose levels or increasing dosing frequency of aTNFα on top of an already-established CIC and cCIC response did not lead to substantial changes in CIC, cCIC formation, or IC deposition. Finally, no association between the presence of CICs or cCIC in plasma and glomerular IC deposition and/or glomerulonephritis was observed.

Formation, clearance, deposition, pathogenicity, and identification of biopharmaceutical-related immune complexes: review and case studies.

Vascular inflammation, infusion reactions, glomerulopathies, and other potentially adverse effects may be observed in laboratory animals, including monkeys, on toxicity studies of therapeutic monoclonal antibodies and recombinant human protein drugs. Histopathologic and immunohistochemical (IHC) evaluation suggests these effects may be mediated by deposition of immune complexes (ICs) containing the drug, endogenous immunoglobulin, and/or complement components in the affected tissues. ICs may be observed in glomerulus, blood vessels, synovium, lung, liver, skin, eye, choroid plexus, or other tissues or bound to neutrophils, monocytes/macrophages, or platelets. IC deposition may activate complement, kinin, and/or coagulation/fibrinolytic pathways and result in a systemic proinflammatory response. IC clearance is biphasic in humans and monkeys (first from plasma to liver and/or spleen, second from liver or spleen). IC deposition/clearance is affected by IC composition, immunomodulation, and/or complement activation. Case studies are presented from toxicity study monkeys or rats and indicate IHC-IC deposition patterns similar to those predicted by experimental studies of IC-mediated reactions to heterologous protein administration to monkeys and other species. The IHC-staining patterns are consistent with findings associated with generalized and localized IC-associated pathology in humans. However, manifestations of immunogenicity in preclinical species are generally not considered predictive to humans.

Immune complex disease in a chronic monkey study with a humanised, therapeutic antibody against CCL20 is associated with complement-containing drug aggregates.

Despite the potential for the chemokine class as therapeutic targets in immune mediated disease, success has been limited. Many chemokines can bind to multiple receptors and many receptors have multiple ligands, with few exceptions. One of those exceptions is CCL20, which exclusively pairs to CCR6 and is associated with several immunologic conditions, thus providing a promising therapeutic target. Following successful evaluation in a single dose, first time in human clinical study, GSK3050002-a humanized IgG1 monoclonal antibody against human CCL20-was evaluated in a 26-week cynomolgus monkey toxicology study. A high incidence of unexpected vascular and organ inflammation was observed microscopically, leading to the decision to halt clinical development. Here we report a dose-responsive increase in the incidence and severity of inflammation in multiple organs from monkeys receiving 30 and 300 mg/kg/week by either subcutaneous or intravenous injection. Histomorphological changes resembled an immune complex-mediated pathology, which is often due to formation of anti-drug antibodies in monkeys receiving a human protein therapeutic and thus not predictive of clinical outcome. However, the presentation was atypical in that there was a clear dose response with a very high incidence of inflammation with a low incidence of ADA that did not correlate well individually. Additionally, the immunohistologic presentation was atypical in that the severity and distribution of tissue inflammation was greater than the numbers of associated immune complexes (i.e., granular deposits). An extensive ex vivo analysis of large molecular weight protein complexes in monkey serum from this study and in human serum samples demonstrated a time-dependent aggregation of GSK3050002, that was not predicted by in vitro assays. The aggregates also contained complement components. These findings support the hypothesis that immune complexes of drug aggregates, not necessarily including anti-drug antibodies, can fix complement, accumulate over time, and trigger immune complex disease. A situation which may have increased clinical relevance than typical anti-drug antibody-associated immune complex disease in monkeys administered human antibody proteins.

Formation and glomerular deposition of immune complexes in mice administered bovine serum albumin: Evaluation of dose, frequency, and biomarkers.

In preclinical toxicity studies, species-foreign proteins administered to animals frequently leads to formation of anti-drug antibodies (ADA). Such antibodies may form circulating immune complexes (CIC) with the administered protein. These CIC can activate the classical complement pathway, thereby forming complement-bound CIC (cCIC); if large of amounts of CIC or cCIC is formed, the clearance mechanism may become saturated which potentially leads to vascular immune complex (IC) deposition and inflammation. Limited information is available on the effect of different treatment related procedures as well as biomarkers of IC-related vascular disease. In order to explore the effect of different dose regimens on IC formation and deposition, and identification of possible biomarkers of IC deposition and IC-related pathological changes, C57BL/6J and BALB/c mice were dosed subcutaneously twice weekly with bovine serum albumin (BSA) for 13 weeks without adjuvant. After 6 and 13 weeks, CIC and cCIC were detected in plasma; after 13 weeks, IC deposition was detected in kidney glomeruli. In particular immunohistochemistry double-staining was shown to be useful for detection of IC deposition. Increasing dosing frequency or changing BSA dose level on top of an already established CIC and cCIC response did not cause changes in IC deposition, but CIC and cCIC concentrations tended to decrease with increased dose level, and increased cCIC formation was observed after more frequent dosing. The presence of CIC in plasma was associated with glomerular IC deposits in the dose regimen study; however, the use of CIC or cCIC as potential biomarkers for IC deposition and IC-related pathological changes, needs to be explored further.

Amyloidosis in the black-footed ferret (Mustela nigripes).

This study describes clinical, histologic, immunohistochemical and electron microscopic features of amyloid A amyloidosis occurring in black-footed ferrets (Mustela nigripes) from eight U.S. zoological institutions. Ferrets had nonregenerative anemia, serum chemistries consistent with chronic renal disease, and proteinuria. Amyloid was present in a variety of tissues, but it was most severe in renal glomeruli and associated with tubular protein loss and emaciation. Congo red/potassium permanganate (KMnO4) and immunohistochemical stains revealed that the amyloid was of the AA type. Concurrent diseases and genetic predisposition were considered the most important contributing factors to development of amyloidosis. Analysis of the genetic tree did not reveal convincing evidence of a common ancestor in the affected ferrets, but a genetic predisposition is likely because all the captive black-footed ferrets are related.

High incidence of lymphoid neoplasia in a colony of Egyptian spiny-tailed lizards (Uromastyx aegyptius).

Hematopoietic malignancies are the most commonly reported neoplasms in lizards, occurring sporadically as in other reptiles. An unusually high incidence of lymphoid neoplasia occurred in a collection of Egyptian spiny-tailed lizards (Uromastyx aegyptius) from 1993-2001. Eight of 15 lizards necropsied at the Louisville Zoological Garden (53%) had multicentric lymphoma. Immunohistochemistry was not useful in characterizing the lineage of normal or neoplastic lymphocytes. By light and electron microscopy (EM), the neoplasms had plasmacytoid morphologic features suggesting B-cell origin, although some tumors also had a primitive lymphoblast component. A concurrent leukemic blood profile was identified in seven of the cases (88%). All were adult animals and no sex predilection was observed. No exposure to exogenous carcinogens was observed. Some of the lizards were unrelated, so hereditary factors were unlikely. Although examination by EM and viral isolation performed on archived tissues and plasma failed to detect viruses, an infectious etiology still warrants consideration.

Inclusion body myositis in spring peepers (Pseudacris crucifer).

In 2000, 2 adult captive spring peepers (Pseudacris crucifer) from the same zoological park were humanely euthanized. Histologically, both frogs had degeneration, atrophy, and necrosis of striated myofibers of the tongue admixed with chronic lymphohistiocytic inflammation. One frog had similar lesions in the skeletal muscles of the body wall. Several degenerate and necrotic myofibers contained single, eosinophilic, intranuclear inclusion bodies. Ultrastructural examination of the inclusions revealed nonenveloped, icosahedral, virus-like particles averaging 20-24 nm in diameter. This is the first reported case of inclusion body myositis in frogs and is believed to be due to parvoviral infection.

Degenerative encephalopathy in a coastal mountain kingsnake (Lampropeltis zonata multifasciata) due to adenoviral-like infection.

In March 2000, an approximately 30-yr-old, male coastal mountain kingsnake (Lampropeltis zonata multifasciata) presented with disequilibrium and unresponsiveness to stimuli that ultimately lead to euthanasia. Histologically, there were foci of gliosis primarily within the caudal cerebrum, brainstem, and cervical spinal cord. Several glial cells and endothelial cells contained magenta, intranuclear inclusion bodies. Electron microscopy of the inclusions revealed paracrystalline arrays of 79-82 nm, viral-like particles. DNA in situ hybridization of sections of formalin-fixed brain using a mixture of two digoxigenin-end-labeled, adenovirus specific, oligonucleotide probes at low and high stringency was positive for adenovirus.

Reptile neoplasia: a retrospective study of case submissions to a specialty diagnostic service.

This retrospective study appears to be the largest publication to date regarding the prevalence of neoplasia in reptiles. As in previous publications, neoplasia is most common in snakes, followed by lizards, chelonians, and crocodilians. Several interesting trends were documented in this study, some of which appear to be previously unidentified, and some that support the findings of previous publications.

Causes of mortality in captive Attwater's prairie chickens (Tympanuchus cupido attwateri) at the San Antonio Zoo, 1997-2000.

Necropsy records for 102 Attwater's prairie chickens (Tympanuchus cupido attwateri) that had been captive-reared and had died at the San Antonio Zoo over a 4-yr period were evaluated to determine the causes of mortality. Gross necropsy, histopathology, and bacterial culture findings are summarized. Mortality was highest in young birds (<6 mo) and embryos. Husbandry- and gastrointestinal-related diseases were the most common causes of mortality.

Pancreatic islet fibrosis in rock hyrax (Procavia capensis), Part 2: Pathology, immunohistochemistry, and electron microscopy.

Pancreatic islet fibrosis with varying degrees of islet cell hyperplasia or islet effacement was diagnosed histologically in 19 rock hyraxes (Procavia capensis) from seven zoological parks. Some, but not all, affected hyraxes were from a common lineage. The condition was associated with apparent hyperglycemia in seven and diabetes mellitus in two. Immunohistochemistry revealed hyperplasia of beta, alpha, and delta cells proportional to the degree of the fibrosis. Electron microscopy revealed collagen deposition and fibroplasia within and around the islets. Special stains and electron microscopy were negative for the presence of amyloid. Beta cell depletion was never identified. The condition has morphologic features that resemble islet fibrosis of human infants born to diabetic mothers.

Congenital diffuse hyperplastic goiter associated with perinatal mortality in 11 captive-born bottlenose dolphins (Tursiops truncatus).

Diffuse hyperplastic goiter was diagnosed by histopathology in 11 perinatal bottlenose dolphins (Tursiops truncatus) that died at four separate zoos and aquaria. Thyroid morphology of these animals was compared with the histologically normal thyroids of two stranded wild bottlenose dolphin calves, a neonate and a 2-mo-old calf. Histologic changes included reduced follicular luminal diameter, markedly reduced or absent luminal colloid, hypertrophy of follicular epithelium, and follicular dysplasia. The etiology of the thyroid gland lesion was not identified. Cause of death was not determined for most of these animals, but they were presumed to have died from metabolic derangements associated with the thyroid lesion, drowning, or dystocia.