Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80/CD86-CD28 costimulatory pathway

The development of recombinant techniques for rapid cloning, expression, an d characterization of cDNAs encoding antibody (Ab) subunits has revolutioni zed the field of antibody engineering. By fusion to heterologous protein do mains, chain shuffling, or inclusion of self-assembly motifs, novel molecul es such as bispecific Abs can be generated that possess the subset of funct ional 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 transp lanted organs. We designed single-chain Fv antibodies, monospecific and bis pecific diabodies, and a bispecific tetravalent anti body (BiTAb) molecule directed against the CD80 and/or CD86 costimulatory molecules. These recomb inant Ab molecules were expressed in Escherichia coli, followed by purifica tion and evaluation for specific inter action 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. (C) 2001 Academic Press.