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.

Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding.

Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. The sole high-affinity IgG receptor, FcγRI plays a significant role in immunotherapy. To elucidate the molecular mechanism of its high-affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcγRI in complex with the Fc domain of human IgG1.

Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies.

Interleukin-21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4+ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies.

Crystal structure of Fcγ receptor I and its implication in high affinity γ-immunoglobulin binding.

Fcγ receptors (FcγRs) play critical roles in humoral and cellular immune responses through interactions with the Fc region of immunoglobulin G (IgG). Among them, FcγRI is the only high affinity receptor for IgG and thus is a potential target for immunotherapy. Here we report the first crystal structure of an FcγRI with all three extracellular Ig-like domains (designated as D1, D2, and D3).

B-lymphocyte stimulator/a proliferation-inducing ligand heterotrimers are elevated in the sera of patients with autoimmune disease and are neutralized by atacicept and B-cell maturation antigen-immunoglobulin.

Introduction: B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) are members of the tumor necrosis factor (TNF) family that regulate B-cell maturation, survival, and function. They are overexpressed in a variety of autoimmune diseases and reportedly exist in vivo not only as homotrimers, but also as BLyS/APRIL heterotrimers.

Methods: A proprietary N-terminal trimerization domain was used to produce recombinant BLyS/APRIL heterotrimers.

Generation of a high-affinity Fcgamma receptor by Ig-domain swapping between human CD64A and CD16A.

A recombinant soluble version of the human high-affinity receptor for IgG, rh-FcgammaRIA or CD64A, was expressed in mammalian cells and purified from their conditioned media. As assessed by circular dichroism, size exclusion chromatography and dynamic light scattering, incubation of rh-FcgammaRIA at 37 degrees C resulted in time-dependent formation of soluble aggregates caused by protein unfolding and loss of native structure. Aggregate formation was irreversible, temperature-dependent and was independent of rh-FcgammaRIA concentration.

A dual-targeting PDGFRbeta/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo.

Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VEGF and PDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics.

Engineering of stable bispecific antibodies targeting IL-17A and IL-23.

Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic.

Recombinant soluble human FcgammaR1A (CD64A) reduces inflammation in murine collagen-induced arthritis.

Binding of immune complexes to cellular FcgammaRs can promote cell activation and inflammation. In previous studies, a recombinant human (rh) soluble FcgammaR, rh-FcgammaRIA (CD64A), was shown to block inflammation in passive transfer models of immune complex-mediated disease. To assess whether rh-FcgammaRIA could block inflammation in a T cell- and B cell-dependent model of immune complex-mediated disease, the efficacy of rh-FcgammaRIA in collagen-induced arthritis was evaluated.

Targeting immune complex-mediated hypersensitivity with recombinant soluble human FcgammaRIA (CD64A).

Binding of Ag-Ab immune complexes to cellular FcgammaR promotes cell activation, release of inflammatory mediators, and tissue destruction characteristic of autoimmune disease. To evaluate whether a soluble FcgammaR could block the proinflammatory effects of immune complexes, recombinant human (rh) versions of FcgammaRIA, FcgammaRIIA, and FcgammaRIIIA were prepared. Binding of rh-FcgammaRIA to IgG was of high affinity (KD=1.