Immunoglobulin G Fc-binding peptides and their applications

Abstract

{Part 1: IgG Fc binding ligands and their use in antibody research} Rapidly increasing application of antibodies has inspired the development of several novel methods to capture or fix antibodies using smart (bio)materials. The Fc-domain of antibodies is the primary binding site for secondary antibodies and effector proteins among others, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulins and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of bacterial proteins, which can invoke an immune response in the host. Many research groups are actively developing synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are used to fix antibodies on solid surfaces and to increase the half-life of proteins in serum. In this dissertation, recent developments of Fc-binding ligands are presented and their binding characteristics and diverse applications are discussed.

{Part II: Cyclic peptide ligand for antibody purification} The increasing implementation of antibodies in therapeutic and diagnostic applications necessitates the development of antibody production and purification technologies for both, academic and industrial usage. Bacterial Protein A and Protein G are known to bind antibodies with high affinity and have facilitated the isolation and purification thereof. Recently, small peptide ligands (i.e. IgG Fc-domain binding peptide, Fc-III, FcBP) that specifically bind to the Fc-domain of antibodies with high affinity were reported. In the present study we report the development of a reusable high affinity column for antibody purification using Sepharose immobilized FcBP, a disulfide bridged peptide comprising 13 amino acid residues. In addition to FcBP, Cys to Ser substituted FcBP (FcBP-Ser), reduced FcBP (FcBP-Red), commercial Protein A, and Protein G resins were packed into columns and evaluated for antibody purification. The columns were loaded with FcBP, reduced FcBP (FcBP-Red), protein A, and protein G showed good binding capacity for trastuzumab and cetuximab. The column packed with FcBP-Red allowed antibody elution at a less acidic pH (pH 4.8) than is required for the other affinity ligand columns used in our experiments (i.e., pH 3.2 for Protein G and FcBP columns, and pH 3.5 for Protein A). The use of the FcBP column allowed antibody isolation from swine serum in a purity of 95\%, which is the same level of purity obtained using theProtein A and Protein G columns. Interestingly, the FcBP column could be easily regenerated and operated without loss of efficiency for up to 60 runs, the maximum number of runs investigated in the present study.

f{Part III: Antibody delivery into live cells using a IgG Fc-binding peptide conjugated to Tat} While the use of RNA interference results in the total loss of target proteins, antibodies can bind to the corresponding epitope region without affecting the expression level of the target protein. Consequently, antibodies may offer the opportunity to selectively evaluate the biological function of selected domains from multi-domain proteins where each domain has in its unique biological function. Although several methods for antibody transduction into live cells are known, most of them suffer from harsh conditions or low delivery efficiency. In this study, we developed an antibody transducer by conjugating a IgG Fc-binding peptide (FcBP) with Tat, a cell penetrating peptide (CPP). The prepared FcBP-CPP fusion peptide and FcBP-eGFP-Tat fusion protein readily bind to IgGs \textit{in situ} upon simple mixing in cell growth media, and are subsequently translocated into the cytosol of live cells. Since FcBP-CPP reversibly binds to the Fc-region of antibodies, it neither impairs the structural and functional integrity of the antibodies nor requires additional effort to modify the antibodies. This strategy represents enables antibody delivery into live cells in the absence of any additional detergents or other physical treatments such as electrophoresis or microinjection.