Zenobia Therapeutics (United States)

Myeloid cells can restrain antitumor immunity by metabolic pathways, such as the degradation of l-arginine, whose concentrations are regulated by the arginase 1 (ARG1) enzyme. Results from preclinical studies indicate the important role of arginine metabolism in pancreatic ductal adenocarcinoma (PDAC) progression, suggesting a potential for clinical application; however, divergent evolution in ARG1 expression and function in rodents and humans has restricted clinical translation. To overcome this dichotomy, here, we show that neutrophil extracellular traps (NETs), released by spontaneously activated neutrophils isolated from patients with PDAC, create a microdomain where cathepsin S (CTSS) cleaves human (h)ARG1 into different molecular forms endowed with enhanced enzymatic activity at physiological pH. NET-associated hARG1 suppresses T lymphocytes whose proliferation is restored by either adding a hARG1-specific monoclonal antibody (mAb) or preventing CTSS-mediated cleavage, whereas small-molecule inhibitors are not effective. We show that ARG1 blockade, combined with immune checkpoint inhibitors, can restore CD8+ T cell function in ex vivo PDAC tumors. Furthermore, anti-hARG1 mAbs increase the frequency of adoptively transferred tumor-specific CD8+ T cells in tumor and enhance the effectiveness of immune checkpoint therapy in humanized mice. Thus, this study shows that extracellular ARG1, released by activated myeloid cells, localizes in NETs, where it interacts with CTSS that in turn cleaves ARG1, producing major molecular forms endowed with different enzymatic activity at physiological pH. Once exocytosed, ARG1 activity can be targeted by mAbs, which bear potential for clinical application for the treatment of PDAC and require further exploration.

The neonatal crystallizable fragment receptor (FcRn) is responsible for maintaining the long half-life and high levels of the two most abundant circulating proteins, albumin and IgG. In the latter case, the protective mechanism derives from FcRn binding to IgG in the weakly acidic environment contained within endosomes of hematopoietic and parenchymal cells, whereupon IgG is diverted from degradation in lysosomes and is recycled. The cellular location and mechanism by which FcRn protects albumin are partially understood. Here we demonstrate that mice with global or liver-specific FcRn deletion exhibit hypoalbuminemia, albumin loss into the bile, and increased albumin levels in the hepatocyte. In vitro models with polarized cells illustrate that FcRn mediates basal recycling and bidirectional transcytosis of albumin and uniquely determines the physiologic release of newly synthesized albumin into the basal milieu. These properties allow hepatic FcRn to mediate albumin delivery and maintenance in the circulation, but they also enhance sensitivity to the albumin-bound hepatotoxin, acetaminophen (APAP). As such, global or liver-specific deletion of FcRn results in resistance to APAP-induced liver injury through increased albumin loss into the bile and increased intracellular albumin scavenging of reactive oxygen species. Further, protection from injury is achieved by pharmacologic blockade of FcRn-albumin interactions with monoclonal antibodies or peptide mimetics, which cause hypoalbuminemia, biliary loss of albumin, and increased intracellular accumulation of albumin in the hepatocyte. Together, these studies demonstrate that the main function of hepatic FcRn is to direct albumin into the circulation, thereby also increasing hepatocyte sensitivity to toxicity.

Crystallographic fragment screening is a technique for initiating drug discovery in which protein crystals are soaked or grown with high concentrations of small molecule compounds (typically MW 110-250 Da) chosen to represent fragments of potential drugs. Specific binding of these compounds to the protein is subsequently visualized in electron density maps obtained from analysis of X-ray diffraction data collected from these crystals. Theoretical and practical experience indicate that a suitably diverse library of fragment compounds containing only a few hundred compounds may be sufficient to provide a comprehensive screen of the protein target. By soaking crystals in mixtures of 3-10 compounds a fragment screen may be completed within ∼100 diffraction data sets. This data collection requirement may be met given reproducible well-diffracting protein crystals and robotic sample handling equipment at a high flux X-ray source. The leading practical issue for most crystallography laboratories that wish to launch a fragment screening project is the design and/or procurement of an appropriate fragment library. Although several off-the-shelf fragment libraries are available from chemical suppliers, the numbers, sizes, and solubility of the compounds in relatively few of these libraries are well-match to the specific needs of the crystallographic screening experiment. Informed consideration of the properties of compounds in the screening library, possibly augmented by additional filtering of available compounds with appropriate search tools, is required to design a successful experiment. The analysis of results from crystallographic fragment screening involves highly repetitive application of routine image data processing and structure refinement calculations from many very similar crystals. Efficient handling of the data applies a high-throughput structure determination methodology that conveniently packages the structure solution calculations into a single process that provides the crystallographer-analyst with ready-to-view maps for evaluating crystals for bound compounds.