ABSTRACT
This corrects the article DOI: 10.1038/nature24042.
ABSTRACT
Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these 'non-homeostatic' conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the 'emergency circuit' that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.
Subject(s)
Body Weight/physiology , Brain Stem/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Growth Differentiation Factor 15/metabolism , Animals , Brain Stem/cytology , Brain Stem/drug effects , Central Amygdaloid Nucleus/cytology , Central Amygdaloid Nucleus/physiology , Eating/physiology , Energy Metabolism/physiology , Feeding Behavior , Female , Glial Cell Line-Derived Neurotrophic Factor Receptors/deficiency , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Growth Differentiation Factor 15/genetics , Growth Differentiation Factor 15/pharmacology , Homeostasis , Male , Mice , Mice, Knockout , Neurons/drug effects , Neurons/metabolism , Parabrachial Nucleus/cytology , Parabrachial Nucleus/physiology , Stress, PsychologicalABSTRACT
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels approximately 2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR(-/-) mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.
Subject(s)
Antibodies, Monoclonal/immunology , Cholesterol/blood , Neutralization Tests , Serine Endopeptidases/immunology , Animals , Cholesterol/immunology , Crystallography, X-Ray , Macaca fascicularis , Mice , Mice, Inbred C57BL , Mice, Knockout , Proprotein Convertase 9 , Proprotein Convertases , Receptors, LDL/genetics , Receptors, LDL/physiologyABSTRACT
Human IgG2 exists as a mixture of disulfide-linked structural isoforms that can show different activities. To probe the contribution of specific cysteine residues to the formation of structural isoforms, we characterized a series of Cys-->Ser mutant IgG2 recombinant monoclonal antibodies, focused on the first C(H)1 cysteine and the first two hinge cysteines. These residues participate in the formation of structural isoforms that have been noted by nonreduced capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, and cation exchange chromatography. We show that single Cys-->Ser mutants can greatly reduce heterogeneous disulfide bonding in human IgG2 and maintain in vitro activity. The data demonstrate the feasibility of applying site-directed mutagenesis to reduce disulfide bond heterogeneity in human IgG2 while preserving the activity of this therapeutically important class of human antibodies.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Disulfides/chemistry , Immunoglobulin G/chemistry , Immunoglobulin G/genetics , Mutagenesis, Site-Directed , Amino Acid Substitution/genetics , Antibodies, Monoclonal/metabolism , Cysteine/genetics , Cysteine/metabolism , Disulfides/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Immunoglobulin G/metabolism , Immunoglobulin kappa-Chains/chemistry , Immunoglobulin kappa-Chains/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Mapping , Recombinant Proteins/chemistry , Recombinant Proteins/classification , Recombinant Proteins/metabolism , Serine/genetics , Serine/metabolism , Spectrometry, Mass, Electrospray IonizationABSTRACT
An appropriate assessment strategy with validated anti-drug antibody (ADA) assays is critical for comparative evaluation of immunogenicity between a proposed biosimilar and its reference product. The strategy should aim to identify potential differences in immune responses between these products. While an ADA assay employing the proposed biosimilar product as the detecting reagent has been generally recommended for such evaluation, a product-specific assay using the product of interest may be of use as it offers a capability of detecting antibodies against specific epitopes from the respective product. Regardless of assay strategy, the performance of the assay must be fully assessed and method needs to be validated to meet the comparative purpose of immunogenicity assessment.
Subject(s)
Biosimilar Pharmaceuticals/pharmacology , Drug Discovery/methods , Immunity/drug effects , Animals , HumansABSTRACT
The ICOS (Inducible T cell Co-Stimulator)/B7RP-1 (B7-related protein 1) interaction is critical for the proper activation of a T lymphocyte. In this manuscript we describe a systematic in vivo approach to determine the level of blockade required to impair the generation of a T cell-dependent antibody response. We have developed an overall strategy for correlating drug exposure, target saturation, and efficacy in a biological response that can be generalized for most protein therapeutics. Using this strategy, we determined that low levels of B7RP-1 blockade are still sufficient to inhibit the immune response. These data suggest that contact between the T cell and the antigen-presenting cell during antigen presentation is much more sensitive to inhibition than previously believed and that ICOS/B7RP-1 blockade may be efficacious in the treatment of autoimmune diseases.