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1.
MAbs ; 7(4): 743-51, 2015.
Article in English | MEDLINE | ID: mdl-25970007

ABSTRACT

Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.


Subject(s)
Antibody-Dependent Cell Cytotoxicity/drug effects , Antigens, CD/immunology , Immunoglobulin G , Leukocytes/immunology , Phagocytosis/drug effects , Receptors, Fc/immunology , Animals , Antibody-Dependent Cell Cytotoxicity/immunology , Female , HEK293 Cells , Humans , Immunoglobulin A/immunology , Immunoglobulin A/pharmacology , Immunoglobulin G/immunology , Immunoglobulin G/pharmacology , Male , Mice , Mice, Inbred BALB C , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-2/immunology , Receptors, IgG/immunology
2.
J Cell Sci ; 117(Pt 3): 373-80, 2004 Jan 26.
Article in English | MEDLINE | ID: mdl-14702383

ABSTRACT

CD44 is a widely distributed type I transmembrane glycoprotein and functions as the major hyaluronan receptor on most cell types. Although alternative splicing can produce a large number of different isoforms, they all retain the hyaluronan-binding Link-homology region and a common transmembrane and cytoplasmic domain, which are highly conserved between species. The past decade has seen an extensive investigation of this receptor owing to its importance in mediating cell-cell and cell-matrix interactions in both normal and disease states. Although roles for alternative splicing and variable glycosylation in determining ligand-binding interactions are now well established, the mechanisms by which CD44 integrates structural and signalling events to elicit cellular responses have been less well understood. However, there is now increasing evidence that CD44 is assembled in a regulated manner into membrane-cytoskeletal junctional complexes and, through both direct and indirect interactions, serves to focus downstream signal transduction events.


Subject(s)
Cell Adhesion/physiology , Hyaluronan Receptors/metabolism , Signal Transduction/physiology , Alternative Splicing/genetics , Amino Acid Sequence , Animals , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Membrane/metabolism , Glycosylation , Humans , Hyaluronan Receptors/genetics , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Isoforms/metabolism , Protein Structure, Tertiary , Sequence Homology, Amino Acid
3.
Nat Cell Biol ; 4(6): 399-407, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12032545

ABSTRACT

The dynamic assembly and disassembly of membrane cytoskeleton junctional complexes is critical in cell migration. Here we describe a novel phosphorylation mechanism that regulates the hyaluronan receptor CD44. In resting cells, CD44 is constitutively phosphorylated at a single serine residue, Ser325. After protein kinase C is activated, a switch in phosphorylation results in CD44 being phosphorylated solely at an alternative residue, Ser291. Using fluorescence resonance energy transfer (FRET) monitored by fluorescence lifetime imaging microscopy (FLIM) and chemotaxis assays we show that phosphorylation of Ser291 modulates the interaction between CD44 and the cytoskeletal linker protein ezrin in vivo, and that this phosphorylation is critical for CD44-dependent directional cell motility.


Subject(s)
Cell Movement/physiology , Hyaluronan Receptors/metabolism , Phosphoproteins/metabolism , Protein Kinase C/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Cytoskeletal Proteins , Humans , Hyaluronan Receptors/immunology , Melanoma , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Phorbol Esters/pharmacology , Phosphorylation/drug effects , Serine/metabolism , Tumor Cells, Cultured
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