Winston Evering

Combination of 4-1BB Agonist and PD-1 Antagonist Promotes Antitumor Effector/Memory CD8 T Cells in a Poorly Immunogenic Tumor Model

Chen, Lee, and colleagues compared the antitumor activity of anti-PD-1 in combination with anti-4-1BB versus with anti-LAG-3 and showed in syngeneic, poorly immunogenic mouse tumor models that the combination with anti-4-1BB elicited superior and well-tolerated tumor inhibition that did not require vaccine. Immunotherapies targeting the programmed death 1 (PD-1) coinhibitory receptor have shown great promise for a subset of patients with cancer. However, robust and safe combination therapies are still needed to bring the benefit of cancer immunotherapy to broader patient populations. To search for an optimal strategy of combinatorial immunotherapy, we have compared the antitumor activity of the anti–4-1BB/anti–PD-1 combination with that of the anti–PD-1/anti–LAG-3 combination in the poorly immunogenic B16F10 melanoma model. Pronounced tumor inhibition occurred only in animals receiving anti–PD-1 and anti–4-1BB concomitantly, while combining anti–PD-1 with anti–LAG-3 led to a modest degree of tumor suppression. The activity of the anti–4-1BB/anti–PD-1 combination was dependent on IFNγ and CD8+ T cells. Both 4-1BB and PD-1 proteins were elevated on the surface of CD8+ T cells by anti–4-1BB/anti–PD-1 cotreatment. In the tumor microenvironment, an effective antitumor immune response was induced as indicated by the increased CD8+/Treg ratio and the enrichment of genes such as Cd3e, Cd8a, Ifng, and Eomes. In the spleen, the combination treatment shaped the immune system to an effector/memory phenotype and increased the overall activity of tumor-specific CD8+ CTLs, reflecting a long-lasting systemic antitumor response. Furthermore, combination treatment in C57BL/6 mice showed no additional safety signals, and only minimally increased severity of the known toxicity relative to 4-1BB agonist alone. Therefore, in the absence of any cancer vaccine, anti–4-1BB/anti–PD-1 combination therapy is sufficient to elicit a robust antitumor effector/memory T-cell response in an aggressive tumor model and is therefore a candidate for combination trials in patients. Cancer Immunol Res; 3(2); 149–60. ©2014 AACR.

Nonclinical Safety Evaluation of Sunitinib: A Potent Inhibitor of VEGF, PDGF, KIT, FLT3, and RET Receptors

Sunitinib malate (SUTENT) is a multitargeted receptor tyrosine kinase (RTK) inhibitor that is approved multinationally for the treatment of imatinib-resistant/-intolerant gastrointestinal stromal tumor and advanced renal cell carcinoma. This paper characterizes the organ toxicity of sunitinib in Sprague-Dawley rats and cynomolgus monkeys, and the reversibility of any treatment-induced effects. Rats and monkeys received sunitinib (0–15 and 0–20 mg/kg/day, respectively) orally on a consecutive daily dosing schedule for thirteen weeks or on an intermittent daily dosing schedule for up to nine months. Clinical observations and laboratory parameters were recorded. Necropsy was conducted following treatment/recovery periods, and histologic examinations were performed. In rats, sunitinib was generally tolerated at 0.3 and 1.5 mg/kg/day, and findings were reversible. In monkeys, the level at which there were no observed adverse effects was 1.5 mg/kg/day, and findings were similarly reversible (except for uterine/ovarian weight changes and skin pallor). Data suggest that inhibition of multiple RTK pathways may induce pharmacologic effects on organ systems in nonclinical species. Key pharmacologic effects of sunitinib included reversible inhibition of neovascularization into the epiphyseal growth plate, and impaired corpora lutea formation and uterine development during estrus. Similar observations have been noted with this class of RTK signaling inhibitors and are consistent with pharmacologic perturbations of physiologic/angiogenic processes associated with the intended molecular targets.

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.

Differentiating Spontaneous from Drug-Induced Vascular Injury in the Dog

When vascular injury is observed in dogs used in preclinical toxicology studies, careful evaluation of the lesions is warranted, especially when differentiating drug-induced vascular changes from spontaneous findings, such as idiopathic canine polyarteritis. The clinical signs as well as the nature and distribution of lesions can often be distinguishing, as is the case with vasoactive drugs, including vasodilators and/or positive inotropes (hydralazine, minoxidil, endothelin receptor antagonists, and phosphodiesterase III inhibitors). For most types of vasodilator-induced vascular injury, the lesion is often restricted to coronary arteries, whereas in idiopathic canine polyarteritis, arterial lesions not only involve coronary arteries, but also medium to small arteries of other organs. In addition, the nature of the changes in vessels yields important clues. Medial and adventitial hemorrhage is generally associated with vasodilator-induced arterial lesion, whereas hemorrhage is generally absent in idiopathic polyarteritis. Although idiopathic canine polyarteritis can generally be differentiated from vasoactive-induced vascular injury in dogs, there are increasing incidences of this type of polyarteritis in dogs receiving any 1 of a number of unrelated classes of compounds, suggestive of an exacerbation of the spontaneous disease. Therefore, in order to differentiate drug-induced injury from idiopathic canine polyarteritis, it is critical that examination of the vascular pathology be conducted with good understanding of clinical, pharmacological, and mechanistic data associated with the drug.

miR-208a as a Biomarker of Isoproterenol-induced Cardiac Injury in Sod2+/− and C57BL/6J Wild-type Mice

This investigation examined microRNA-208a (miR-208a) as a potential biomarker of isoproterenol (ISO)-induced cardiac injury in superoxide dismutase-2 (Sod2+/− ) and the wild-type mice, and the potential sensitivity of Sod2+/− mice to ISO-induced toxicity. A single intraperitoneal injection of ISO was administered to age-matched wild-type and Sod2+/− mice at 0, 80, or 160 mg/kg. Plasma miR-208a, cardiac troponin I (cTnI), and ISO systemic exposure were measured at various time points postdose. Hearts were collected for histopathology examination and for tissue expression of miR-208a and myosin heavy chain 7. ISO administration caused increases in cTnI and miR-208a plasma levels that correlated with myocardial damage; however, the magnitude of increase differed according to the types of mice. At similar ISO systemic exposure, the magnitude of cTnI was greater in wild-type mice compared to Sod2+/ − mice; however, the magnitude of miR-208a was greater in Sod2+/− mice than that of the wild-type mice. Myocardial degeneration occurred at ≥3 hr in the wild-type and ≥6 hr in Sod2+/ − mice. At ≥24 hr after ISO administration, miR-208a appeared superior to cTnI in indicating myocardial injury in both wild-type and Sod2+/− mice. Sod2+/− mice were not more sensitive than wild-type mice to ISO-induced toxicity.

Epithelial Tissue Hyperplasia Induced by the RAF Inhibitor PF-04880594 is Attenuated by a Clinically Well-Tolerated Dose of the MEK Inhibitor PD-0325901

Clinical trials of selective RAF inhibitors in patients with melanoma tumors harboring activated BRAFV600E have produced very promising results, and a RAF inhibitor has been approved for treatment of advanced melanoma. However, about a third of patients developed resectable skin tumors during the course of trials. This is likely related to observations that RAF inhibitors activate extracellular signal–regulated kinase (ERK) signaling, stimulate proliferation, and induce epithelial hyperplasia in preclinical models. Because these findings raise safety concerns about RAF inhibitor development, we further investigated the underlying mechanisms. We showed that the RAF inhibitor PF-04880594 induces ERK phosphorylation and RAF dimerization in those epithelial tissues that undergo hyperplasia. Hyperplasia and ERK hyperphosphorylation are prevented by treatment with the mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor PD-0325901 at exposures that extrapolate to clinically well-tolerated doses. To facilitate mechanistic and toxicologic studies, we developed a three-dimensional cell culture model of epithelial layering that recapitulated the RAF inhibitor–induced hyperplasia and reversal by MEK inhibitor in vitro. We also showed that PF-04880594 stimulates production of the inflammatory cytokine interleukin 8 in HL-60 cells, suggesting a possible mechanism for the skin flushing observed in dogs. The complete inhibition of hyperplasia by MEK inhibitor in epithelial tissues does not seem to reduce RAF inhibitor efficacy and, in fact, allows doubling of the PF-04880594 dose without toxicity usually associated with such doses. These findings indicated that combination treatment with MEK inhibitors might greatly increase the safety and therapeutic index of RAF inhibitors for the treatment of melanoma and other cancers. Mol Cancer Ther; 11(10); 2274–83. ©2012 AACR.

Evaluation of miR-216a and miR-217 as Potential Biomarkers of Acute Exocrine Pancreatic Toxicity in Rats:

Detecting and monitoring exocrine pancreatic damage during nonclinical and clinical testing is challenging because classical biomarkers amylase and lipase have limited sensitivity and specificity. Novel biomarkers for drug-induced pancreatic injury are needed to improve safety assessment and reduce late-stage attrition rates. In a series of studies, miR-216a and miR-217 were evaluated as potential biomarkers of acute exocrine pancreatic toxicity in rats. Our results revealed that miR-216a and miR-217 were almost exclusively expressed in rat pancreas and that circulating miR-216a and miR-217 were significantly increased in rats following administration of established exocrine pancreatic toxicants caerulein (CL) and 1-cyano-2-hydroxy-3-butene (CHB) as well as in rats administered a proprietary molecule known to primarily affect the exocrine pancreas. Conversely, neither microRNA was increased in rats administered a proprietary molecule known to cause a lesion at the pancreatic endocrine–exocrine interface (EEI) or in rats administered an established renal toxicant. Compared with amylase and lipase, increases in miR-216a and miR-217 were of greater magnitude, persisted longer, and/or correlated better with microscopic findings within the exocrine pancreas. Our findings demonstrate that in rats, miR-216a and miR-217 are sensitive and specific biomarkers of acute exocrine pancreatic toxicity that may add value to the measurement of classical pancreatic biomarkers.

Lack of Respiratory and Contact Sensitizing Potential of the Intranasal Antiviral Drug Candidate Rupintrivir (AG7088): A Weight-of-the-Evidence Evaluation

Rupintrivir, also known as AG7088, is a small molecule 3C protease inhibitor designed to target human rhinovirus as a potential intranasal treatment for the common cold. The ability of rupintrivir to induce both respiratory and contact hypersensitivity responses was evaluated using a weight of the evidence approach. A local lymph node assay (LLNA) in mice evaluating concentrations of rupintrivir up to 50% in dimethylformamide showed no evidence of sensitizing capability. An irritation study conducted in rabbits was performed to assess potential dermal irritancy and provide information for worker safety guidelines. The study showed no evidence of skin irritation when the material was placed in direct contact with the skin in a semi-occluded fashion for four days. Quantitative whole body autoradiography (QWBA) following intranasal instillation of the compound into rabbits showed that the compound was retained in the nasal cavity or was swallowed. No radioactivity was observed in the pulmonary regions of these animals. Histopathologic evaluation of the nasopharyngeal tract and the lungs of both rats and dogs exposed by intranasal instillation acutely or following repeated intranasal exposures for 14 (rat) or 28 days (rat and dog) did not reveal any evidence of irritation or inflammation in these regions of the respiratory tract. These data demonstrate that rupintrivir does not cause irritation or inflammatory responses that may precede the development of sensitization of the skin or respiratory tract. It was concluded that the weight of the available toxicologic evidence indicated that rupintrivir was not likely to cause sensitization of either the skin or the respiratory tract in humans following intranasal delivery.

Comparison of two decalcification agents using microwave technology: a histochemical assessment of the rat cochlea

ABSTRACT Despite the prevalence of hearing loss, there are no FDA-approved chemical entities available for either otoprotection or treatment of hearing impairment. This lack of drug-based treatments has made the assessment of the middle and inner ear a rare, challenging exercise in the pharmaceutical industry. Recently, advances in the use of various decalcifying agents, as well as heating techniques utilizing microwave technology have proven to be effective at shortening processing times, minimizing tissue damage and potentially lowering the cost of histopathological evaluation and diagnosis. This study tested decalcification agents in order to determine the most efficient decalcification procedure for rat outer, middle, and inner ear structures while preserving tissue morphology and eliminating introduction of artifacts to best facilitate histopathological analysis. Presented here are the results of rat ear histological preparations using two decalcifying agents at increasing microwave times with heat controlled at the physiological temperature of 37 °C. In this analysis, an acid-based combination decalcification-fixative solution was found to significantly speed up the decalcification process when compared with the calcium chelator EDTA.

CDK4/6 Inhibition on Glucose and Pancreatic Beta Cell Homeostasis in Young and Aged Rats

Genetic deletion of cyclin-dependent kinase 4 (Cdk4) is associated with pancreatic beta cell loss and glucose dysregulation in rodents. Palbociclib, one of the first selective CDK4/6 inhibitors approved for the treatment of advanced breast cancer, is currently being investigated as an adjuvant treatment in patients with early-stage breast cancer and in a variety of cancers covering a wide-range of patient populations. Hence, longer chronic toxicity studies were necessary to further examine its safety profile. The effects of different doses and duration of palbociclib administration on glucose and beta cell homeostasis in young (two months) versus aged (12 months) rats was compared. Glucose dysregulation, due to pancreatic beta cell degeneration, was observed in young rats administered the highest dose of palbociclib for 6 months. Abnormal pancreatic islet histology and activation of the endoplasmic reticulum stress response in beta cells were detected after shorter administration with high-dose palbociclib in young rats. To test the hypothesis that palbociclib-associated inhibition of beta cell proliferation will more profoundly affect younger animals that have not achieved replicative quiescence, we administered high-dose palbociclib to aged rats for 6 months. In contrast to the young rats, despite equivalent exposures to palbociclib, no evidence of impaired glucose tolerance, hypoinsulinemia, beta cell vacuolization, or beta cell loss was seen in aged rats. Palbociclib administration induces beta cell failure in young but not aged rats. Implications: Although adult humans receiving palbociclib have not displayed detectable adverse effects on glucose metabolism, the risk of beta cell failure in children remains unexplored. Mol Cancer Res; 15(11); 1531–41. ©2017 AACR.