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.

Crystal structure and receptor-interacting residues of MYDGF - a protein mediating ischemic tissue repair.

Myeloid-derived growth factor (MYDGF) is a paracrine-acting protein that is produced by bone marrow-derived monocytes and macrophages to protect and repair the heart after myocardial infarction (MI). This effect can be used for the development of protein-based therapies for ischemic tissue repair, also beyond the sole application in heart tissue. Here, we report the X-ray structure of MYDGF and identify its functionally relevant receptor binding epitope. MYDGF consists of a 10-stranded ß-sandwich with a folding topology showing no similarities to other cytokines or growth factors. By characterizing the epitope of a neutralizing antibody and utilizing functional assays to study the activity of surface patch-mutations, we were able to localize the receptor interaction interface to a region around two surface tyrosine residues 71 and 73 and an adjacent prominent loop structure of residues 97-101. These findings enable structure-guided protein engineering to develop modified MYDGF variants with potentially improved properties for clinical use.

Plasma Concentrations of Myeloid-Derived Growth Factor in Healthy Individuals and Patients with Acute Myocardial Infarction as Assessed by Multiple Reaction Monitoring-Mass Spectrometry.

Myeloid-derived growth factor (MYDGF in humans, Mydgf in mice) is a secreted protein with previously unknown biological functions. In a recent study, Mydgf was shown to mediate cardiac repair after acute myocardial infarction (MI) in mice. Lack of a sensitive assay to measure MYDGF in the circulation has hampered its further investigation. Here, we developed a liquid chromatography/multiple reaction monitoring-mass spectrometry MYDGF assay, employing SDS-PAGE-based protein fractionation to deplete high-abundant proteins and a stable isotope-labeled synthetic standard peptide for quantification. The assay had a lower limit of quantification of 0.8 ng/mL and a linear range up to 190 ng/mL. Within-run and total imprecision ranged from 8 to 17% and 11 to 20%, respectively. MYDGF plasma concentrations were not affected by either storage at room temperature for 4 h or up to three freeze-thaw cycles. Apparently healthy adults presented with a median (range) MYDGF concentration of 3.3 (1.3-6.7) ng/mL ( n = 120). MYDGF concentrations were elevated 2.7-fold ( P < 0.001) in patients with acute MI ( n = 101) and were associated with inflammatory biomarkers, renal dysfunction, and long-term cardiovascular mortality. The new assay and the favorable preanalytic characteristics of the analyte will facilitate studies into the pathophysiology of MYDGF and its potential use as a biomarker or protein therapeutic in patients with acute MI or other disease states.

Optimizing NBE PK/PD assays using the Gyrolab Affinity Software; conveniently within the bioanalyst's existing workflow.

AIM: The fully automated microfluidics-based Gyrolab is a popular instrument for the bioanalysis of protein therapeutics; requiring minimal sample and reagent volumes. Gyros offers affinity software for determining binding affinity in solution using a high-throughput method and miniaturized reactions. RESULTS: Using this affinity software, multiple CTGF-targeting reagents were characterized on the Gyrolab after <100% target coverage was seen in a cynomolgus pharmacokinetic/PD study dosed with anti-CTGF antibodies. The results uncovered magnitude differences in binding affinities between the dosed antibody, target and assay reagents. CONCLUSION: The binding affinity values were used to investigate reduced target coverage and results highlight potential of the affinity software for incorporation into the bioanalyst's existing Gyrolab workflow for characterizing reagents and optimizing pharmacokinetic/PD bioanalytical assays.