Mapping the Energetic Epitope of an Antibody/Interleukin-23 Interaction with Hydrogen/Deuterium Exchange, Fast Photochemical Oxidation of Proteins Mass Spectrometry, and Alanine Shave Mutagenesis.

Epitope mapping the specific residues of an antibody/antigen interaction can be used to support mechanistic interpretation, antibody optimization, and epitope novelty assessment. Thus, there is a strong need for mapping methods, particularly integrative ones. Here, we report the identification of an energetic epitope by determining the interfacial hot-spot that dominates the binding affinity for an anti-interleukin-23 (anti-IL-23) antibody by using the complementary approaches of hydrogen/deuterium exchange mass spectrometry (HDX-MS), fast photochemical oxidation of proteins (FPOP), alanine shave mutagenesis, and binding analytics.

The structure of the death receptor 4-TNF-related apoptosis-inducing ligand (DR4-TRAIL) complex.

The structure of death receptor 4 (DR4) in complex with TNF-related apoptosis-inducing ligand (TRAIL) has been determined at 3 Å resolution and compared with those of previously determined DR5-TRAIL complexes. Consistent with the high sequence similarity between DR4 and DR5, the overall arrangement of the DR4-TRAIL complex does not differ substantially from that of the DR5-TRAIL complex. However, subtle differences are apparent.

Integrated Pharmacokinetic/Pharmacodynamic Analysis for Determining the Minimal Anticipated Biological Effect Level of a Novel Anti-CD28 Receptor Antagonist BMS-931699.

BMS-931699 (lulizumab pegol), a domain antibody (dAb) conjugated with 40-kDa branched polyethylene glycol, is a human anti-CD28 receptor antagonist under development for the treatment of inflammatory and autoimmune diseases. In the present work, the minimal anticipated biologic effect level (MABEL) was determined for BMS-931699 by integrating all the available preclinical data. The relevance of the in vitro mixed lymphocyte reaction (MLR) assay to a whole blood CD28 receptor occupancy (RO) assessment, as well as the relationship between the CD28 RO and the inhibition of T-cell-dependent antibody response to keyhole limpet hemocyanin in vivo, was demonstrated through an integrated pharmacokinetic/pharmacodynamic analysis using anti-hCD28 dAb-001 (differing from BMS-931699 by two additional amino acids at the N-terminus) and a mouse surrogate.

Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody.

Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy).

2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses.

Mounting evidence in models of both autoimmunity and chronic viral infection suggests that the outcome of T cell activation is critically impacted by the constellation of co-stimulatory and co-inhibitory receptors expressed on the cell surface. Here, we identified a critical role for the co-inhibitory SLAM family member 2B4 (CD244) in attenuating primary antigen-specific CD8(+) T cell responses in the presence of immune modulation with selective CD28 blockade. Our results reveal a specific up-regulation of 2B4 on antigen-specific CD8(+) T cells in animals in which CD28 signaling was blocked.

Antibody-unfolding and metastable-state binding in force spectroscopy and recognition imaging.

Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds.

The structure of apolipoprotein A-II in discoidal high density lipoproteins.

It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available.

Functional dissection of osteoprotegerin and its interaction with receptor activator of NF-kappaB ligand.

The receptor activator of NF-kappaB (RANK) belongs to the neuregulin/tumor necrosis factor (TNF) receptor superfamily and is activated by RANK ligand (RANK-L), a homotrimeric, TNF-like cytokine. RANK is present on the surface of osteoclast cell precursors, where its interaction with RANK-L induces their terminal differentiation into osteoclasts, thus increasing bone breakdown. The secreted, soluble receptor osteoprotegerin (OPG) interrupts this activation by binding directly to RANK-L.

Structural analysis of lipoprotein E particles.

Apolipoprotein E (apoE) is a key regulator of cholesterol homeostasis. Human apoE has three common isoforms, each with different risk implications for cardiovascular and neurodegenerative disease. Neither the structure of lipoprotein E particles nor the structural consequences of the isoform differences are known.

Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.

Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo.