ABSTRACT
Mast cells (MC) are key drivers of allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 is an immunoregulatory receptor found on MCs. While it is recognized that engaging Siglecs with antibodies mediates inhibition across immune cells, the mechanisms that govern this agonism are not understood. Here we generated Siglec-6 mAb clones (AK01 to AK18) to better understand Siglec-6-mediated agonism. Siglec-6 mAbs displayed epitope-dependent receptor internalization and inhibitory activity. We identified a Siglec-6 mAb (AK04) that required Fc-mediated interaction for receptor internalization and induced inhibition and antibody-dependent cellular phagocytosis against MCs. AK04-mediated MC inhibition required Siglec-6 immunoreceptor tyrosine-based inhibitory motif (ITIM) and ITIM-like domains and was associated with receptor cluster formation containing inhibitory phosphatases. Treatment of humanized mice with AK04 inhibited systemic anaphylaxis with a single dose and reduced MCs with chronic dosing. Our findings suggest Siglec-6 activity is epitope dependent and highlight an agonistic Siglec-6 mAb as a potential therapeutic approach in allergic disease.
Subject(s)
Antigens, CD , Mast Cells , Humans , Mice , Animals , Antigens, CD/chemistry , Sialic Acid Binding Immunoglobulin-like Lectins , Antibodies, Monoclonal/pharmacology , EpitopesABSTRACT
Immunomodulation of mast cell (MC) activity is warranted in allergic and inflammatory diseases where MCs have a central role in pathogenesis. Targeting Siglec-8, an inhibitory receptor on MCs and eosinophils, has shown promising activity in preclinical and clinical studies. While the intracellular pathways that regulate Siglec-8 activity in eosinophils have been well studied, the signaling mechanisms that lead to MC inhibition have not been fully elucidated. Here, we evaluate the intracellular signaling pathways of Siglec-8-mediated inhibition in primary MCs using an anti-Siglec-8 monoclonal antibody (mAb). Phospho-proteomic profiling of FcεRI-activated MCs revealed Siglec-8 mAb-treatment globally inhibited proximal and downstream kinases, leading to attenuated MC activation and degranulation. In fact, Siglec-8 was found to directly interact with FcεRI signaling molecules. Siglec-8 inhibition was dependent on both cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that interact with the SH2 containing protein phosphatase Shp-2 upon Siglec-8 phosphorylation. Taken together, these data support a model in which Siglec-8 regulates proximal FcεRI-induced phosphorylation events through phosphatase recruitment and interaction with FcεRIγ, resulting in global inhibition of MCs upon Siglec-8 mAb engagement.
Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/metabolism , Lectins/metabolism , Mast Cells/immunology , Receptors, IgE/metabolism , Animals , Cell Degranulation , Humans , Mice , Mice, Inbred C57BL , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Proteomics , Signal TransductionABSTRACT
EphA3 is expressed in solid tumors and leukemias and is an attractive target for the therapy. We have generated a panel of Humaneered® antibodies to the ligand-binding domain using a Fab epitope-focused library that has the same specificity as monoclonal antibody mIIIA4. A high-affinity antibody was selected that competes with the mIIIA4 antibody for binding to EphA3 and has an improved affinity of â¼1 nM. In order to generate an antibody with potent cell-killing activity the variable regions were assembled with human IgG1k constant regions and expressed in a Chinese hamster ovary (CHO) cell line deficient in fucosyl transferase. Non-fucosylated antibodies have been reported to have enhanced binding affinity for the IgG receptor CD16a (FcγRIIIa). The affinity of the antibody for recombinant CD16a was enhanced approximately 10-fold. This resulted in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity against EphA3-expressing leukemic cells, providing a potent antibody for the evaluation as a therapeutic agent.
Subject(s)
Antibodies, Monoclonal/immunology , Antibody Affinity , Antibody-Dependent Cell Cytotoxicity , Receptor, EphA3/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , CHO Cells , Cricetinae , Cricetulus , Humans , Immunoglobulin Fc Fragments/immunology , Macaca mulatta , Molecular Sequence Data , Receptors, IgG/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute lung injury and mortality through the delivery of exotoxins by the type III secretion system (TTSS). PcrV is an important structural protein of the TTSS. An engineered human antibody Fab fragment that binds to the P. aeruginosa PcrV protein with high affinity has been identified and has potent in vitro neutralization activity against the TTSS. The instillation of a single dose of Fab into the lungs of mice provided protection against lethal pulmonary challenge of P. aeruginosa and led to a substantial reduction of viable bacterial counts in the lungs. These results demonstrate that blocking of the TTSS by a Fab lacking antibody Fc-mediated effector functions can be sufficient for the effective clearance of pulmonary P. aeruginosa infection.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Toxins/immunology , Immunoglobulin Fab Fragments/immunology , Pore Forming Cytotoxic Proteins/immunology , Pseudomonas Infections/immunology , Recombinant Proteins/immunology , ADP Ribose Transferases/immunology , Animals , Antibody Specificity , Cytotoxicity, Immunologic/immunology , Enzyme-Linked Immunosorbent Assay , Exotoxins/immunology , Humans , Immunoglobulin Fab Fragments/therapeutic use , Male , Mice , Mice, Inbred BALB C , Pseudomonas Infections/prevention & control , Pseudomonas aeruginosa/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/therapeutic use , Virulence Factors/immunology , Pseudomonas aeruginosa Exotoxin AABSTRACT
A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.