Antibody-drug conjugates (ADCs) are a promising therapeutic modality for oncology indications. The concept of an ADC platform is to increase the therapeutic index (TI) of chemotherapeutics through ...more selective delivery of cytotoxic agents to tumor cells while limiting exposure to healthy normal cells. Despite the use of antibodies targeting antigens abundantly and/or exclusively expressed on cancer cells (i.e., target cells), dose limiting toxicities (DLTs) in normal cells/tissues are frequently reported even at suboptimal therapeutic doses. Although advancement of ADC technology has helped to optimize all three key components (i.e., mAb, linker, and payload), DLTs remain a key challenge for ADC development. Mechanisms of ADC toxicity in normal cells/tissues are not clearly understood, but the majority of DLTs are considered to be target-independent. In addition to linker-drug instability contributing to the premature release of cytotoxic drug (payload) in circulation, uptake/trafficking of intact ADCs by both receptor-dependent (FcγRs, FcRn and C-type lectin receptors), and-independent (non-specific endocytosis) mechanisms may contribute to off-target toxicity in normal cells. In this article, we review potential mechanisms of target-independent ADC uptake and toxicity in normal cells, as well as discuss components of ADCs which may influence these mechanisms. This information will provide a deeper understanding of the underlying mechanisms of ADC off-target toxicity and prove helpful toward improving the overall TI of the next generation of ADCs.
Gene expression profiling is a useful tool to predict and interrogate mechanisms of toxicity. RNA-Seq technology has emerged as an attractive alternative to traditional microarray platforms for ...conducting transcriptional profiling. The objective of this work was to compare both transcriptomic platforms to determine whether RNA-Seq offered significant advantages over microarrays for toxicogenomic studies. RNA samples from the livers of rats treated for 5 days with five tool hepatotoxicants (α-naphthylisothiocyanate/ANIT, carbon tetrachloride/CCl
, methylenedianiline/MDA, acetaminophen/APAP, and diclofenac/DCLF) were analyzed with both gene expression platforms (RNA-Seq and microarray). Data were compared to determine any potential added scientific (i.e., better biological or toxicological insight) value offered by RNA-Seq compared to microarrays. RNA-Seq identified more differentially expressed protein-coding genes and provided a wider quantitative range of expression level changes when compared to microarrays. Both platforms identified a larger number of differentially expressed genes (DEGs) in livers of rats treated with ANIT, MDA, and CCl
compared to APAP and DCLF, in agreement with the severity of histopathological findings. Approximately 78% of DEGs identified with microarrays overlapped with RNA-Seq data, with a Spearman's correlation of 0.7 to 0.83. Consistent with the mechanisms of toxicity of ANIT, APAP, MDA and CCl
, both platforms identified dysregulation of liver relevant pathways such as Nrf2, cholesterol biosynthesis, eiF2, hepatic cholestasis, glutathione and LPS/IL-1 mediated RXR inhibition. RNA-Seq data showed additional DEGs that not only significantly enriched these pathways, but also suggested modulation of additional liver relevant pathways. In addition, RNA-Seq enabled the identification of non-coding DEGs that offer a potential for improved mechanistic clarity. Overall, these results indicate that RNA-Seq is an acceptable alternative platform to microarrays for rat toxicogenomic studies with several advantages. Because of its wider dynamic range as well as its ability to identify a larger number of DEGs, RNA-Seq may generate more insight into mechanisms of toxicity. However, more extensive reference data will be necessary to fully leverage these additional RNA-Seq data, especially for non-coding sequences.
Cancer therapies targeting the vascular endothelial growth factor (VEGF) signaling pathway can lead to renal damage by disrupting the glomerular ultrafiltration apparatus. The objective of the ...current study was to identify sensitive biomarkers for VEGF inhibition-induced glomerular changes in rats. Male Sprague-Dawley rats were administered an experimental VEGF receptor (VEGFR) inhibitor, ABT-123, for seven days to investigate the correlation of several biomarkers with microscopic and ultrastructural changes. Glomeruli obtained by laser capture microdissection were also subjected to gene expression analysis to investigate the underlying molecular events of VEGFR inhibition in glomerulus. ABT-123 induced characteristic glomerular ultrastructural changes in rats, including fusion of podocyte foot processes, the presence of subendothelial electron-dense deposits, and swelling and loss of fenestrations in glomerular endothelium. The subtle morphological changes cannot be detected with light microscopy or by changes in standard clinical chemistry and urinalysis. However, urinary albumin increased 44-fold as early as Day three. Urinary β2-microglobulin levels were also increased. Other urinary biomarkers that are typically associated with tubular injury were not significantly impacted. Such patterns in urinary biomarkers can provide valuable diagnostic insight to VEGF inhibition therapy-induced glomeruli injuries.
Microarray technology, which allows one to quantitate the expression of thousands of genes simultaneously, has begun to have a major impact on many different areas of drug discovery and development. ...The question remains of whether microarray analysis and gene expression signature profiles can be applied to the field of toxicology. To date, there are very few published studies showing the use of microarrays in toxicology and important questions remain regarding the predictability and accuracy of applying gene expression profiles to toxicology. To begin to address these questions, we have treated rats with 15 different known hepatotoxins, including allyl alcohol, amiodarone, Aroclor 1254, arsenic, carbamazepine, carbon tetrachloride, diethylnitrosamine, dimethylformamide, diquat, etoposide, indomethacin, methapyrilene, methotrexate, monocrotaline, and 3-methylcholanthrene. These agents cause a variety of hepatocellular injuries including necrosis, DNA damage, cirrhosis, hypertrophy, and hepatic carcinoma. Gene expression analysis was done on RNA from the livers of treated rats and was compared against vehicle-treated controls. The gene expression results were clustered and compared to the histopathology findings and clinical chemistry values. Our results show strong correlation between the histopathology, clinical chemistry, and gene expression profiles induced by the agents. In addition, genes were identified whose regulation correlated strongly with effects on clinical chemistry parameters. Overall, the results suggest that microarray assays may prove to be a highly sensitive technique for safety screening of drug candidates and for the classification of environmental toxins.
A rate-limiting step that occurs in the drug discovery process is toxicological evaluation of new compounds. New techniques that use small amounts of the experimental compound and provide a high ...degree of predictivity would greatly improve this process. The field of microarray technology, which allows one to monitor thousands of gene expression changes simultaneously, is rapidly advancing and is already being applied to numerous areas in toxicology. However, it remains to be determined if compounds with similar toxic mechanisms produce similar changes in transcriptional expression. In addition, it must be determined if gene expression changes caused by an agent in vitro would reflect those produced in vivo. In order to address these questions, we treated rat hepatocytes with 15 known hepatoxins (carbon tetrachloride, allyl alcohol, aroclor 1254, methotrexate, diquat, carbamazepine, methapyrilene, arsenic, diethylnitrosamine, monocrotaline, dimethyl-formamide, amiodarone, indomethacin, etoposide, and 3-methylcholanthrene) and used microarray technology to characterize the compounds based on gene expression changes. Our results showed that gene expressional profiles for compounds with similar toxic mechanisms indeed formed clusters, suggesting a similar effect on transcription. There was not complete identity, however, indicating that each compound produced a unique signature. These results show that large-scale analysis of gene expression using microarray technology has promise as a diagnostic tool for toxicology.
In vitro toxicogenomics represents a useful approach for evaluating the toxic properties of new drug candidates early in the drug discovery process using minimal amounts of compounds. The aim of this ...study was to develop in vitro-based gene expression assays for two prototypical toxicological classes: aryl hydrocarbon receptor (AhR) agonists and peroxisome proliferator activated receptor alpha (PPAR alpha) agonists. Primary rat hepatocytes were exposed to a number of class-specific compounds, including 3-methylcholanthrene, aroclor, and beta-napthoflavone as AhR agonists, bezafibrate, clofibrate, and Wy-14643 as peroxisome proliferators, and chlorpheniramine, penicillin and spectinomycin as negative controls. Global gene expression profiles were generated with microarrays for each class of compounds. Using linear discriminant analysis coupled with permutation-based t-test, gene signatures were established to classify compounds according to a discriminant score. The final gene signatures consist of eight genes for AhR agonism and 11 genes for PPAR alpha agonism, and were further validated using additional compounds. The assay was initially developed using a microarray platform. The authors then evaluated whether it could be transferred to a more cost-effective platform with higher throughput. The results indicate that a small set of genes can be used to quantitatively assess the degree to which a compound falls into a certain mechanistic toxicological class. While this study only focused on two classes, it could be expanded to encompass other toxicological mechanistic classes as well. Furthermore, by adapting this type of assay to a higher throughput platform, in vitro toxicogenomics can represent an effective approach to generate robust toxicological data early in the drug discovery process.
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Drug‐induced biliary toxicity is difficult to monitor
in vivo
and detect
in vitro
and thus remains a confounding issue in drug development. HepaRG cells are an immortalized cell line ...derived from progenitor cells that differentiate into a mixed population of hepatocyte‐like and cholangiocyte‐like (biliary) cells. Leveraging the unique dual biliary/hepatocellular nature of HepaRG cells, we evaluated changes in cellular impedance as an endpoint for distinguishing compounds associated with biliary toxicity. Cryopreserved HepaRG cells (Biopredic) were plated at 30K/well on electrode plates, allowed to mature over 6 days, and treated with various concentrations of prototypical biliary toxicants representing diverse mechanisms (ANIT, cyclosporine, chlorpromazine, carmustine, Bosentan, MDA), known hepatocellular toxicants (acetaminophen (APAP), cadmium chloride) and a benign control compound (penicillin). Cells were monitored for 72 hours on the ACEA xCELLigence real‐time cellular impedance instrument. In parallel, the same treatments were conducted with cryopreserved human hepatocytes (KalyCell). A comparison of profiles showed a greater dose‐dependent decrease in impedance in primary human hepatocytes vs HepaRG regardless of the toxicant class with all but one compound, APAP. Data suggest a reduced basal GSH level in HepaRG cells compared with primary hepatocytes may make these cells more susceptible to APAP toxicity. Based on the conditions and endpoints employed in this study, HepaRG cells provide no clear advantage for either the identification or the mechanistic differentiation of compounds inducing biliary toxicity, but may be more useful for monitoring certain types of hepatotoxicity, particularly those associated with reactive metabolites.
Pharmacokinetic variability in drug plasma exposure between different studies within the same species is not unexpected due to a variety of factors (such as differences in formulation, active ...pharmaceutical ingredient salt form and solid-state, genetic strain, sex, environmental, disease status, bioanalysis methods, circadian rhythms, etc.) although variability from within the same research group typically does not occur to a great degree because these variables are commonly controlled. Surprisingly, a pharmacology proof of concept study with a previously validated tool compound from the literature failed to show expected response in murine glucose-6-phosphate isomerase-induced arthritis model which was tied to compound plasma exposure unexpectedly 10-fold lower than exposure observed from early pharmacokinetic study confirming adequate exposure prior to proof of concept. A systematic series of studies were conducted to investigate causes for exposure difference between pharmacology and pharmacokinetic studies identifying the presence or absence of soy protein in animal chow as the causative variable. Cyp3a11 expression in intestine and liver was determined to increase in a time dependent manner in mice switched to diets containing soybean meal compared with mice on diets without soybean meal. The repeated pharmacology experiments using the soybean meal free diet achieved plasma exposures that were maintained above the EC50 and showed efficacy and proof of concept for the target. This effect was further confirmed with marker CYP3A4 substrates in follow on mouse studies. The role of soy protein containing diets on CYP expression necessitates the inclusion of controlling rodent diet as a variable for preventing possible exposure differences between studies. SIGNIFICANCE STATEMENT: The presence of soybean meal protein in murine diet increased clearance and decreased oral exposure for select cytochrome 3A4 substrates. Related effects were also observed on select liver enzyme expression.
Hydrazine is a rodent carcinogen and is classified as a probable human carcinogen by IARC. Though hydrazine is positive in both in vitro and in vivo DNA strand break (comet) assays, hydrazine was ...reported to be negative in an in vitro mutation Muta Mouse lung epithelial cell (FE1) test, as well as in a regulatory‐compliant, in vivo Big Blue mouse mutation test. In this article, mechanistic studies explored the cellular response to hydrazine. When tested in a regulatory‐compliant mouse lymphoma assay, hydrazine yielded unusual, weakly positive results. This prompted an investigation into the transcriptional response to hydrazine in FE1 cells via RNA sequencing. Amongst the changes identified was a dose‐dependent increase in G2/M DNA damage checkpoint activation associated genes. Flow cytometric experiments in FE1 cells revealed that hydrazine exposure led to S‐phase cell cycle arrest. Clonogenic assays in a variety of cell lines harboring key DNA repair protein deficiencies indicated that hydrazine could sensitize cells lacking homology dependent repair proteins (Brca2 and Fancg). Lastly, hprt assays with hydrazine were conducted to determine whether a lack of DNA repair could lead to mutagenicity. However, no robust, dose‐dependent induction of mutations was noted. The transcriptional and cell cycle response to hydrazine, coupled with functional investigations of DNA repair‐deficient cell lines support the inconsistencies noted in the genetic toxicology regulatory battery. In summary, while hydrazine may be genotoxic, transcriptional and functional processes involved in cell cycle regulation and DNA repair appear to play a nuanced role in mediating the mutagenic potential.
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Antibody‐drug conjugate (ADC) uptake via the mannose receptor or other glycan receptors has been proposed as a possible mechanism of some non‐target‐related toxicities. Mannan, a ...natural polymer of mannose found in yeast cell walls, is serologically similar to structures found on mammalian glycoproteins including immunoglobulins and has been shown to limit the uptake of ADCs in cellular models. Mannan binds to cell surface glycan receptors and is anticipated to alter the tissue distribution of ADCs which can also bind to those receptors via their glycosylation sites. In order to test the tolerability of systemic mannan administration to support its potential use as a mitigating agent that would limit non‐specific ADC uptake, male CD1 mice were dosed up to 5 days with daily IV bolus doses of mannan (from S.cerevisiae) formulated in normal saline at 0, 30, 100, 300 and 1000 mg/kg (n=4/group). All mice in the 30 mg/kg dose group survived and mannan was well tolerated at this dose. Doses of 100 mg/kg and greater were not tolerated and either morbidity or mortality was noted within 1 hour of dosing. The nature of the clinical signs and timing relative to dosing suggest anaphylaxis‐like shock, documented in certain strains of mice with IV mannan administration, or plasma volume expansion as possible causes of death. Histopathological review showed unique retinal changes in only the non‐tolerated dose groups. The changes were characterized by cytoplasmic vacuolation of the inner portion of the inner nuclear layer. Staining for AP‐2 alpha (Activating Enhancer Binding Protein 2 Alpha), an amacrine cell‐specific marker in the inner portion of the inner nuclear layer, confirmed that the vacuolation was indeed restricted to these amacrine cells.
Mannose supplementation has been used in other clinical settings such as for correction of hypoglycosylation resulting from mutations in phosphomannose isomerase (MPI) in congenital disorder of glycosylation. It has been previously reported that female C57BL/6J mice hypomorphic for MPI, that were supplemented with mannose in drinking water at 2–5% during gestation, produced embryos with severe developmental abnormalities resulting in high lethality. Surviving pups showed severe eye defects including an observed decrease in AP‐2 staining in the inner nuclear layer of the retina. Mannose supplementation during adulthood after eye development for 4–6 months in both WT mice and mice hypomorphic for MPI was not associated with similar defects. Differences in route, dose, monomeric vs. polymeric form of mannose and strain of mice could explain why a similar phenotype in retina was observed when mannan was dosed in adult mice in the current study and suggests caution should be observed even in adulthood when mannan supplementation is considered clinically.
Support or Funding Information
RC, SM, CD, MK, WB, TV are employees of Abbvie.
The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
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