Myostatin and activin blockade by engineered follistatin results in hypertrophy and improves dystrophic pathology in mdx mouse more than myostatin blockade alone.

BACKGROUND: Myostatin antagonists are being developed as therapies for Duchenne muscular dystrophy due to their strong hypertrophic effects on skeletal muscle. Engineered follistatin has the potential to combine the hypertrophy of myostatin antagonism with the anti-inflammatory and anti-fibrotic effects of activin A antagonism. METHODS: Engineered follistatin was administered to C57BL/6 mice for 4 weeks, and muscle mass and myofiber size was measured. In the mdx model, engineered follistatin was dosed for 12 weeks in two studies comparing to an Fc fusion of the activin IIB receptor or an anti-myostatin antibody. Functional measurements of grip strength and tetanic force were combined with tissue analysis for markers of necrosis, inflammation, and fibrosis to evaluate improvement in dystrophic pathology. RESULTS: In wild-type and mdx mice, dose-dependent increases in muscle mass and quadriceps myofiber size were observed for engineered follistatin. In mdx, increases in grip strength and tetanic force were combined with improvements in muscle markers for necrosis, inflammation, and fibrosis. Improvements in dystrophic pathology were greater for engineered follistatin than the anti-myostatin antibody. CONCLUSIONS: Engineered follistatin generated hypertrophy and anti-fibrotic effects in the mdx model.

Inhibition of plasma kallikrein by a highly specific active site blocking antibody.

Plasma kallikrein (pKal) proteolytically cleaves high molecular weight kininogen to generate the potent vasodilator and the pro-inflammatory peptide, bradykinin. pKal activity is tightly regulated in healthy individuals by the serpin C1-inhibitor, but individuals with hereditary angioedema (HAE) are deficient in C1-inhibitor and consequently exhibit excessive bradykinin generation that in turn causes debilitating and potentially fatal swelling attacks. To develop a potential therapeutic agent for HAE and other pKal-mediated disorders, we used phage display to discover a fully human IgG1 monoclonal antibody (DX-2930) against pKal. In vitro experiments demonstrated that DX-2930 potently inhibits active pKal (Ki = 0.120 ± 0.005 nM) but does not target either the zymogen (prekallikrein) or any other serine protease tested. These findings are supported by a 2.1-Å resolution crystal structure of pKal complexed to a DX-2930 Fab construct, which establishes that the pKal active site is fully occluded by the antibody. DX-2930 injected subcutaneously into cynomolgus monkeys exhibited a long half-life (t½ ∼ 12.5 days) and blocked high molecular weight kininogen proteolysis in activated plasma in a dose- and time-dependent manner. Furthermore, subcutaneous DX-2930 reduced carrageenan-induced paw edema in rats. A potent and long acting inhibitor of pKal activity could be an effective treatment option for pKal-mediated diseases, such as HAE.

A human monoclonal antibody against insulin-like growth factor-II blocks the growth of human hepatocellular carcinoma cell lines in vitro and in vivo.

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR-directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1-104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (K(D) value of 49 and 10 pmol/L, respectively) compared with IGF-I (approximately 10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor-binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 A. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I-induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A.

Interactions of genistein and related isoflavones with lipid micelles.

Genistein (5,7,4'-trihydroxyisoflavone) modulates the function of several transmembrane ion-channel proteins by mechanisms that are unrelated to phosphorylation events. Daidzein (7,4'-dihydroxy-isoflavone) typically exhibits modest effects, whereas genistin (7-O-glucosyl-genistein) usually exhibits no effect on ion-channel activities. Genistein appears to modulate gramicidin A ion channels by alteration of bilayer mechanical properties, but the associated molecular interactions have not been defined. The incorporation of daidzein into phosphatidylcholine liposomes promotes aggregation and precipitate formation which is problematic for structural studies based on NMR spectroscopy. In the present study, daidzein was incorporated into sodium dodecyl sulfate (SDS) micelles to provide a stable system with no evidence of micelle aggregation. For this reason genistein, daidzein, genistin, and osajin (a diprenyl-genistein derivative) were incorporated into SDS micelles (in D(2)O) to evaluate differences in position and orientation within micelle structures. The (1)H NMR line widths, as a function of Mn(2+) concentration, indicate that genistein is quite mobile and buried within the hydrophobic micelle core. Daidzein and genistin also are mobile but exhibit average positions near the micelle/aqueous interface, with polar groups oriented toward the aqueous compartment. These results demonstrate that daidzein, with only two hydroxyl substituents, has a greater affinity for a polar environment than genistein with three hydroxyl substituents. The 5-hydroxyl group of genistein forms an intramolecular hydrogen bond with the 4-carbonyl group, which diminishes the molecular affinity for a polar matrix. These results suggest an explanation for the relative abilities of these compounds to increase gramicidin channel lifetimes and modulate other ion-channel types.