In vitro characterization of the anti-PD-1 antibody nivolumab, BMS-936558, and in vivo toxicology in non-human primates.

The programmed death-1 (PD-1) receptor serves as an immunologic checkpoint, limiting bystander tissue damage and preventing the development of autoimmunity during inflammatory responses. PD-1 is expressed by activated T cells and downmodulates T-cell effector functions upon binding to its ligands, PD-L1 and PD-L2, on antigen-presenting cells. In patients with cancer, the expression of PD-1 on tumor-infiltrating lymphocytes and its interaction with the ligands on tumor and immune cells in the tumor microenvironment undermine antitumor immunity and support its rationale for PD-1 blockade in cancer immunotherapy. This report details the development and characterization of nivolumab, a fully human IgG4 (S228P) anti-PD-1 receptor-blocking monoclonal antibody. Nivolumab binds to PD-1 with high affinity and specificity, and effectively inhibits the interaction between PD-1 and its ligands. In vitro assays demonstrated the ability of nivolumab to potently enhance T-cell responses and cytokine production in the mixed lymphocyte reaction and superantigen or cytomegalovirus stimulation assays. No in vitro antibody-dependent cell-mediated or complement-dependent cytotoxicity was observed with the use of nivolumab and activated T cells as targets. Nivolumab treatment did not induce adverse immune-related events when given to cynomolgus macaques at high concentrations, independent of circulating anti-nivolumab antibodies where observed. These data provide a comprehensive preclinical characterization of nivolumab, for which antitumor activity and safety have been demonstrated in human clinical trials in various solid tumors.

MicroRNA changes associated with atypical CYP1A1 inducer BMS-764459.

The corticotrophin releasing factor (CRF) receptor I antagonist, BMS-764459 (evaluated as a potential treatment of affective disorders), was orally dosed to female Sprague-Dawley rats once daily for 2 weeks (vehicle control or 175mg/kg/day). To investigate the mechanism of BMS-764459-related liver weight increases, total liver RNA was isolated and evaluated for mRNA gene expression by microarray analysis (assessing the expression of approximately 24,000 genes) from snap-frozen tissue. Subsequently, mRNA and miRNA (microRNA) were also analyzed 5 years later from FFPE (Formalin Fixed Paraffin Embedded) samples via RT-PCR (about 800 miRNA evaluated). Genomic analyses showed that BMS-764459 induces AhR target genes with additional inductions of CYP2B, CYP3A, and Abcc3 consistent with the gene expression pattern of atypical CYP1A1 inducers. Analysis of miRNA expression identified a number of significantly affected miRNAs. To further evaluate their role in atypical CYP1A1 induction, an in silico evaluation of differentially expressed miRNA was performed and their putative mRNA 3'-UTR (untranslated region) binding sequences were evaluated. MiR-680 and miR-29a were identified as potential regulators and biomarkers of atypical CYP1A1 induction by regulating Abcc3, CYP3A and CYP2B as well as a number of AhR targeted genes.

Determination of relative Notch1 and gamma-secretase-related gene expression in puromycin-treated microdissected rat kidneys.

Notch signaling pathways are involved in the regulation of cell differentiation and are highly conserved across species. Notch ligand binding leads to gamma-secretase-mediated proteolytic cleavage of the Notch receptor releasing the Notch intracellular domain, resulting in its subsequent translocation into the nucleus and gene expression regulation. To investigate the level of expression of Notch signaling pathway components in microanatomic regions following renal injury, kidneys from untreated, vehicle control, and puromycin aminonucleoside (PA, 150 mg/kg)-treated rats were evaluated. Frozen tissue sections from rats were microdissected using laser capture microdissection (LCM) to obtain glomeruli, cortical (proximal) tubules, and collecting ducts, and relative gene expression levels of Presenilin1, Notch1 and Hes1 were determined. In untreated rats, the Notch1 expression in glomeruli was higher than in the proximal tubules and similar to that in collecting ducts, whereas Presenilin1 and Hes1 expressions were highest in the collecting ducts, followed by cortical tubules and glomeruli. Following PA-induced renal injury, Hes1 gene expression increased significantly in the glomeruli and tubules compared to the collecting ducts where no injury was observed microscopically. Although these data present some evidence of change in Notch signaling related to injury, the expression of Presenilin1, Notch1, and Hes1 in the microanatomic regions of the kidney following PA treatment were not significantly different when compared to controls. These results demonstrate that there are differences in Notch-related gene expression in the different microanatomic regions of the kidneys in rats and suggest a minimal role for Notch in renal injury induced by PA. In addition, this work shows that LCM coupled with the RT-PCR can be used to determine the relative differences in target gene expression within regions of a complex organ.