Validation of Cross-Species Reactivity of the VEGF-A/PDGFRß Bifunctional Antibody PF-06653157.

PURPOSE: PF-06653157 is a bifunctional antagonist monoclonal antibody (mAb) that targets human VEGF-A ligand and PDGF-Rß. With the advent of PF-06653157 as an angiogenesis inhibitor and potential treatment for angiogenesis deregulation diseases, a relevant toxicology species is needed for toxicity and efficacy studies. Investigative studies were conducted to validate the mAb dual antagonist properties in a human system and determine its cross-reactive pharmacology in nonhuman cells. METHODS: Sequence alignment was used to determine percent sequence identity of VEGF and PDGF receptors and ligands; qualitative reverse transcription polymerase chain reaction (qRT-PCR) was used to determine the presence of PDGF-Rß on cells of interest. The functional activity of PF-06653157 antibody was assessed in human, dog, porcine, rabbit, rat, mouse, and cynomolgus monkey cells treated with VEGF and PDGF ligands through cell proliferation assays and western blot analysis of AKT and p44/p42 (ERK1/2) protein phosphorylation and enzyme-linked immunosorbent assay. RESULTS: PF-06653157 attenuated phosphorylation of AKT and p44/p42 proteins in human and cynomolgus monkey cells. The antibody did not attenuate AKT nor p44/p42 phosphorylation in any other species tested. PDGFR signaling could not be activated with human PDGF ligand in the porcine cells, so PF-06653157 activity in porcine remains inconclusive. CONCLUSION: The PF-06653157 mAb cross-reacts with cynomolgus monkey cells in a similar manner to human cells. Therefore, cynomolgus monkeys are considered the appropriate species for efficacy and regulatory toxicology studies in PF-06653157 development.

The contribution of lysosomotropism to autophagy perturbation.

Autophagy refers to the catabolic process in eukaryotic cells that delivers cytoplasmic material to lysosomes for degradation. This highly conserved process is involved in the clearance of long-lived proteins and damaged organelles. Consequently, autophagy is important in providing nutrients to maintain cellular function under starvation, maintaining cellular homeostasis, and promoting cell survival under certain conditions. Several pathways, including mTOR, have been shown to regulate autophagy. However, the impact of lysosomal function impairment on the autophagy process has not been fully explored. Basic lipophilic compounds can accumulate in lysosomes via pH partitioning leading to perturbation of lysosomal function. Our hypothesis is that these types of compounds can disturb the autophagy process. Eleven drugs previously shown to accumulate in lysosomes were selected and evaluated for their effects on cytotoxicity and autophagy using ATP depletion and LC3 assessment, respectively. All eleven drugs induced increased staining of endogenous LC3 and exogenous GFP-LC3, even at non toxic dose levels. In addition, an increase in the abundance of SQSTM1/p62 by all tested compounds denotes that the increase in LC3 is due to autophagy perturbation rather than enhancement. Furthermore, the gene expression profile resulting from in vitro treatment with these drugs revealed the suppression of plentiful long-lived proteins, including structural cytoskeletal and associated proteins, and extracellular matrix proteins. This finding indicates a retardation of protein turnover which further supports the notion of autophagy inhibition. Interestingly, upregulation of genes containing antioxidant response elements, e.g. glutathione S transferase and NAD(P)H dehydrogenase quinone 1 was observed, suggesting activation of Nrf2 transcription factor. These gene expression changes could be related to an increase in SQSTM1/p62 resulting from autophagy deficiency. In summary, our data indicate that lysosomal accumulation due to the basic lipophilic nature of xenobiotics could be a general mechanism contributing to the perturbation of the autophagy process.