Expression and Purification of Monospecific and Bispecific Recombinant Antibody Fragments Derived from Antibodies That Block the CD80/CD86-CD28 Costimulatory Pathway

• Published in: Protein expression and purification 2001-06, Vol.22 (1), p.11-24
• Main Authors: Dincq, Stéphanie, Bosman, Fons, Buyse, Marie-Ange, Degrieck, Rosina, Celis, Linda, Boer, Marc de, Van Doorsselaere, Veronique, Sablon, Erwin
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Bispecific - chemistry; Antibodies, Bispecific - immunology; Antibodies, Bispecific - isolation & purification; Antibodies, Bispecific - pharmacology; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - isolation & purification; Antibodies, Monoclonal - pharmacology; Antibody Specificity; Antigens, CD - immunology; Antigens, CD - metabolism; B-Lymphocytes - drug effects; B-Lymphocytes - immunology; B-Lymphocytes - metabolism; B7-1 Antigen - immunology; B7-1 Antigen - metabolism; B7-2 Antigen; Binding Sites, Antibody; Blotting, Western; CD28 Antigens - immunology; CD28 Antigens - metabolism; Cell Line; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Immune Tolerance - drug effects; Immune Tolerance - immunology; Immunoglobulin Fragments - chemistry; Immunoglobulin Fragments - immunology; Immunoglobulin Fragments - isolation & purification; Immunoglobulin Fragments - pharmacology; Immunoglobulin Variable Region - chemistry; Immunoglobulin Variable Region - immunology; Immunoglobulin Variable Region - isolation & purification; Immunoglobulin Variable Region - pharmacology; Membrane Glycoproteins - immunology; Membrane Glycoproteins - metabolism; Mice; Models, Molecular; Protein Engineering; Recombinant Proteins - chemistry; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Recombinant Proteins - pharmacology; Signal Transduction - drug effects
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.2001.1417

The development of recombinant techniques for rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, or inclusion of self-assembly motifs, novel molecules such as bispecific Abs can be generated that possess the subset of functional properties designed to fit the intended application. We describe the engineering of Ab fragments produced in bacteria for blocking the CD28-CD80/CD86 costimulatory interaction in order to induce tolerance against transplanted organs. We designed single-chain Fv antibodies, monospecific and bispecific diabodies, and a bispecific tetravalent antibody (BiTAb) molecule directed against the CD80 and/or CD86 costimulatory molecules. These recombinant Ab molecules were expressed in Escherichia coli, followed by purification and evaluation for specific interaction with their respective antigen in an enzyme-linked immunosorbent assay (ELISA). A specific sandwich ELISA confirmed the bispecificity of the bispecific diabodies and the BiTAb.