Development of novel ultra-sensitive IL-11 target engagement assays to support mechanistic PK/PD modeling for an anti-IL-11 antibody therapeutic.

An in-house antibody generation campaign identified a diverse, high affinity set of anti-interleukin-11 (IL-11) monoclonal antibodies (mAbs) to enable successful development of novel, custom ultra-sensitive target engagement assays for detection of "free" (unbound to the dosed anti-IL-11 therapeutic mAb) and "total" (free and mAb-IL-11 complexed form) IL-11 in preclinical species and human. Antibody hits from distinct epitope communities were screened on various platforms, including enzyme-linked immunosorbent assay, Meso Scale Discovery, Simoa HD-1 and Simoa Planar Array (SP-X), and used for assay development and sensitivity optimization. The ultra-sensitive SP-X format achieved a lower limit of quantitation of 0.006 pg/mL, enabling the first reported baseline levels of IL-11 in healthy control plasma determined by custom bioanalytical assays. These newly established baseline levels supported mechanistic pharmacokinetic/pharmacodynamic modeling in mouse, cynomolgus monkey, and human for a greater understanding of preclinical study design and in vivo dynamic interaction of soluble IL-11 with an anti-IL-11 antibody therapeutic candidate. Modeling and simulation also helped refine the utility of assays with respect to their potential use as target engagement biomarkers in the clinic.Abbreviations IL-11: Interleukin-11, TE: Target engagement, PK/PD: Pharmacokinetic/pharmacodynamic, mAb: Monoclonal antibody, NHP: Non-human primate, IgG: Immunoglobulin G, Cyno: Cynomolgulus monkey, GFR: Glomerular filtration rate, BQL: Below quantitation levels, DRM: Disease relevant model, kDa: kilodaltons, SPR: Surface plasmon resonance, pSTAT3: phosphorylated STAT3, IL-11R: Interleukin-11 receptor, TPP: Target product protein, LLOQ: Lower limit of quantitation, RLU: Relative light units.

Effect of Novel Biotherapeutic Elevating Angiopoietin 1 on Progression of Diabetic Nephropathy in Diabetic/Obese Mice.

Diabetic nephropathy is a leading cause of end-stage renal disease, characterized by endothelial dysfunction and a compromised glomerular permeability barrier. Dysregulation of the angiopoietin 1 (ANGPT1)/angiopoietin 2 (ANGPT2) signaling axis is implicated in disease progression. We recently described the discovery of an IgG1 antibody, O010, with therapeutic potential to elevate circulating endogenous ANGPT1, a tyrosine kinase with Ig and epidermal growth factor (EGF) homology domains-2 (TIE2) agonist. Studies are described that detail the effect of various ANGPT1-elevating strategies to limit progression of renal dysfunction in diabetic-obese (db/db) mice. Results demonstrate that adeno-associated virus- or DNA minicircle-directed overexpression of ANGPT1 elicits a reduction in albuminuria (56%-73%) and an improvement in histopathology score (18% reduction in glomerulosclerosis). An improved acetylcholine response in isolated aortic rings was also observed indicative of a benefit on vascular function. In separate pharmacokinetic studies, an efficacious dose of the ANGPT1 DNA minicircle increased circulating levels of the protein by >80%, resulting in a concomitant suppression of ANGPT2. At a dose of O010-producing maximal elevation of circulating ANGPT1 achievable with the molecule (60% increase), no suppression of ANGPT2 was observed in db/db mice, suggesting insufficient pathway engagement; no reduction in albuminuria or improvement in histopathological outcomes were observed. To pinpoint the mechanism resulting in lack of efficacy, we demonstrate, using confocal microscopy, an interference with TIE2 translocation to adherens junctions, resulting in a loss of protection against vascular permeability normally conferred by ANGPT1. Results demonstrated the essential importance of ANGPT1 to maintain the glomerular permeability barrier, and, due to interference of O010 with this process, led to the discontinuation of the molecule for clinical development. SIGNIFICANCE STATEMENT: This body of original research demonstrates that elevation of systemic angiopoietin 1 (ANGPT1) is protective against diabetic nephropathy. However, using a novel biotherapeutic approach to elevate systemic ANGPT1 renoprotection was not observed; we demonstrate that protection was lost due to interference of the therapeutic with ANGPT1/ tyrosine kinase with Ig and EGF homology domains-2 translocation to adherens junctions. Thus, the clinical development of the antibody was terminated.

Chemical Derivatization Enables MALDI-TOF-Based High-Throughput Screening for Microbial Trimethylamine (TMA)-Lyase Inhibitors.

Microbial-dependent trimethylamine (TMA) generation from dietary precursors such as choline was recently linked to cardiovascular diseases (CVDs) as well as chronic kidney disease (CKD). Inhibition of TMA-generating enzymes in gut bacteria would be an innovative approach to treat these diseases. The potential to accurately quantify secreted TMA levels highlights the capacity of mass spectrometry (MS) for tracking microbial TMA-lyase activity. However, high-throughput screening (HTS) by conventional MS instrumentation is hampered by limited sample throughput. Recent advancement in liquid handling and instrumentation of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS provides an HTS-compatible MS technology. The deciphering of enzymatic reactions using this label-free readout has been successfully applied but has thus far been limited to peptide/protein-centric activity assays. Here, we demonstrate the versatile applicability of MALDI-TOF by tracking a small molecule within a highly complex sample background. The key to success for this concept was chemical derivatization of the target molecule enabling quantitative assessment of microbial TMA formation. Further, its potential was demonstrated in a side-by-side comparison to RapidFire-MS in a primary screen and subsequent dose-response experiments. Overall, the established assay enables the screening for microbial TMA-lyase inhibitors and serves as a proof of concept for the applicability of MALDI-TOF for demanding assay concepts per se.

Targeting VE-PTP phosphatase protects the kidney from diabetic injury.

Diabetic nephropathy is a leading cause of end-stage kidney failure. Reduced angiopoietin-TIE2 receptor tyrosine kinase signaling in the vasculature leads to increased vascular permeability, inflammation, and endothelial cell loss and is associated with the development of diabetic complications. Here, we identified a mechanism to explain how TIE2 signaling is attenuated in diabetic animals. Expression of vascular endothelial protein tyrosine phosphatase VE-PTP (also known as PTPRB), which dephosphorylates TIE2, is robustly up-regulated in the renal microvasculature of diabetic rodents, thereby reducing TIE2 activity. Increased VE-PTP expression was dependent on hypoxia-inducible factor transcriptional activity in vivo. Genetic deletion of VE-PTP restored TIE2 activity independent of ligand availability and protected kidney structure and function in a mouse model of severe diabetic nephropathy. Mechanistically, inhibition of VE-PTP activated endothelial nitric oxide synthase and led to nuclear exclusion of the FOXO1 transcription factor, reducing expression of pro-inflammatory and pro-fibrotic gene targets. In sum, we identify inhibition of VE-PTP as a promising therapeutic target to protect the kidney from diabetic injury.

Glomerular mesangial cell recruitment and function require the co-receptor neuropilin-1.

Proteinuria has been reported in cancer patients receiving agents that target the transmembrane receptor neuropilin-1 (Nrp1) suggesting potential adverse effects on glomerular function. Here we show that Nrp1 is highly expressed by mesangial cells and that genetic deletion of the Nrp1 gene from PDGF receptor-ß+ mesangial cells results in proteinuric disease and glomerulosclerosis, leading to renal failure and death within 6 wk of age in mice. The major defect is a failure of mesangial cell migration that is required to establish the mature glomerular tuft. In vitro data show that the potent chemotactic effect of PDGFB is lost in Nrp1-deficient mesangial cells. Biochemical analyses reveal that Nrp1 is required for PDGFB-dependent phosphorylation of p130 Crk-associated substrate (p130Cas), a large-scaffold molecule that is involved in motility of other cell types. In stark contrast, matrix adhesion and activation of ERK and Akt, which mediate proliferation of mesangial cells in response to PDGFB, are unaffected by the absence of Nrp1. Taken together, these results identify a critical cell-autonomous role for Nrp1 in the migratory behavior of mesangial cells and may help explain the renal effects that occur in patients receiving Nrp1-inhibitory drugs.

Vascular Growth Factors and Glomerular Disease.

The glomerulus is a highly specialized microvascular bed that filters blood to form primary urinary filtrate. It contains four cell types: fenestrated endothelial cells, specialized vascular support cells termed podocytes, perivascular mesangial cells, and parietal epithelial cells. Glomerular cell-cell communication is critical for the development and maintenance of the glomerular filtration barrier. VEGF, ANGPT, EGF, SEMA3A, TGF-ß, and CXCL12 signal in paracrine fashions between the podocytes, endothelium, and mesangium associated with the glomerular capillary bed to maintain filtration barrier function. In this review, we summarize the current understanding of these signaling pathways in the development and maintenance of the glomerulus and the progression of disease.

EP1 disruption attenuates end-organ damage in a mouse model of hypertension.

Prostaglandin E(2) is a major prostanoid found in the kidney and vasculature contributing to the regulation of blood pressure. The prostaglandin E(2) receptor EP1 has been shown to contribute to hypertension by mediating angiotensin II-dependent vasoconstriction, although its precise role is incompletely characterized. Disruption of the EP1 receptor in C57BL/6J mice reduced the incidence of mortality during severe hypertension induced by uninephrectomy, deoxycorticosterone acetate, and angiotensin II. Mortality was dependent on all components of the model. Death was a result of aortic aneurysm rupture or occurred after development of anasarca, each of which was reduced in EP1-/- mice. Mean arterial pressure was increased in treated EP1+/+ and EP1-/- mice; however, this elevation was significantly lower in EP1-/- mice. Blood pressure reduction via administration of hydralazine phenocopied EP1-/- mice. Thus, reduction in blood pressure by disruption of EP1 reduced incidence of mortality and decreased organ damage, suggesting that EP1 receptor blockade may be a viable target for antihypertensive therapy.