ABSTRACT
Methods for identifying and producing cell specific affinity reagents are critical in cell detection, separation, and therapeutic delivery applications, yet remain difficult and time consuming. To address these limitations, a rapid and quantitative screening approach was developed using intrinsically fluorescent bacterial display peptide libraries and fluorescence-activated cell sorting (FACS). High-throughput screening of fluorescent libraries yielded a panel of peptide ligands mediating specific recognition of human breast cancer tumor cells. Clonal populations of fluorescent, peptide-displaying bacteria enabled single-step, fluorescent labeling of the target cells for cytometry and microscopy analysis. Isolated peptides could be categorized into several distinct groups possessing strong consensus sequences with as many as six identities. Importantly, individual clones exhibited high specificity target cell binding, with more than 80-fold increased binding to tumor cells (ZR-75-1) relative to cell lines derived from healthy tissue (HMEC, MCF-10A). Fluorescent display libraries thus provide a powerful new methodology for parallel identification of cell specific affinity ligands.
Subject(s)
Peptide Library , Affinity Labels , Amino Acid Sequence , Breast/cytology , Breast/metabolism , Breast Neoplasms/metabolism , Cell Line, Tumor , Cells, Cultured , Escherichia coli/genetics , Female , Green Fluorescent Proteins/genetics , Humans , Ligands , Microscopy, Fluorescence , Molecular Sequence Data , Peptides/genetics , Peptides/metabolismABSTRACT
PURPOSE OF REVIEW: This review summarizes current knowledge regarding the control of human mast cell and basophil signaling and recent developments using a new therapeutic platform consisting of a human bifunctional gamma and epsilon heavy chain (Fcgamma-Fcepsilon) protein to inhibit allergic reactivity. RECENT FINDINGS: Crosslinking of FcgammaRIIb to FcepsilonRI on human mast cells and basophils by a genetically engineered Fcgamma-Fcepsilon protein (GE2) leads to the inhibition of mediator release upon FcepsilonRI challenge. GE2 protein was shown to inhibit cord blood-derived mast cell and peripheral blood basophil mediator release in vitro in a dose dependent fashion including inhibition of human IgE reactivity to cat. In addition, IgE-mediated release from lung tissue was inhibited through GE2. The mechanism of inhibition in mast cells included alterations in IgE-mediated Ca2+ mobilization, Syk phosphorylation and the formation of Dok-Grb2-SHIP complex. Proallergic effects of Langerhans-like dendritic cells and B cell IgE switching were also inhibited by GE2. In vivo, GE2 was shown to block passive cutaneous anaphylaxis (PCA) driven by human IgE in mice expressing the human FcepsilonRI and inhibit skin test reactivity to dust mite antigen in a dose dependent manner in rhesus monkeys. The balance between positive and negative signaling controls mast cell and basophil reactivity that is critical in the expression of human allergic diseases. This approach using a human Fcgamma-Fcepsilon fusion protein to co-aggregate FcepsilonRI with the FcgammaRII holds promise as a new therapeutic platform for the immunomodulation of allergic diseases and potentially other mast cell/basophil-dependent disease states.
Subject(s)
Basophils/physiology , Hypersensitivity/therapy , Mast Cells/physiology , Receptors, IgE/therapeutic use , Receptors, IgG/therapeutic use , Recombinant Fusion Proteins/therapeutic use , Signal Transduction/physiology , B-Lymphocytes/physiology , Dendritic Cells/physiology , Humans , Hypersensitivity/immunologyABSTRACT
Variable region domain exchanged IgG, or "inside-out (io)," molecules, were produced to investigate the effects of domain interactions on antibody structure and function. Studies using ultracentrifugation and electron microscopy showed that variable region domain exchange induces non-covalent multimerization through Fab domains. Surprisingly, variable region exchange also affected Fc-associated functions such as serum half-life and binding to protein G and FcgammaRI. These alterations were not merely a consequence of IgG aggregation. Both the extent of multimerization and alterations in Fc-associated properties depended on the IgG isotype.
Subject(s)
Antigen-Antibody Complex , Immunoglobulin G/immunology , Immunoglobulin Variable Region/immunology , Dimerization , Humans , Immunoglobulin Fab Fragments , Immunoglobulin Fc Fragments , Immunoglobulin G/chemistry , Immunoglobulin G/metabolism , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/metabolism , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Structure, Tertiary , Receptors, IgG/metabolism , Recombinant Fusion ProteinsABSTRACT
Unlike IgA and IgM, IgG has not yet been shown to form covalent polymers. However in the presence of specific Ag, murine IgG3 has been shown to polymerize through noncovalent interactions. In contrast to the noncovalent oligomers found with murine IgG3, we have detected covalent dimers in three different recombinant human IgG2 Abs produced in myeloma cells. Both IgG2,kappa and IgG2,lambda can form dimers. In addition, analysis of pooled human gamma globulin and several normal sera revealed the presence of IgG2 dimers. The IgG2 dimers are in contrast to the noncovalent IgG dimers found in pooled sera of multiple donors resulting from idiotype/anti-idiotype (Id/anti-Id) interactions. Cyanogen bromide cleavage analysis suggests that one or more Cys residues in the gamma 2 hinge are involved in dimer assembly. The potential role of IgG2 dimers in immunity against carbohydrate Ags is discussed.
