title: Binding affinity and inhibitory properties of a single-chain anti-T cell receptor antibody.
abstract: The antigen-specific function of T cells is mediated through a surface receptor (alpha beta). Antibodies to the receptor can either inhibit or activate the T cell depending on the multimeric form of the antibody and the isotype of the antibody constant regions. To further examine and eventually control these antagonistic properties, an anti-receptor antibody (1B2) was engineered by linking the variable region genes of the heavy and light chains to form a single-chain binding domain (scFv). The native 1B2 antibody recognizes determinants on the variable regions of both the alpha and beta subunits of the T cell receptor. The scFv gene was expressed in Escherichia coli, and after folding by dilution of solubilized inclusion bodies, scFv protein was purified as both a 29,000-Da monomer and a noncovalently associated 54,000-Da dimer. The binding affinity of the monomer (KD = 17 nM) was 20-fold lower than that of 1B2 Fab fragments (KD = 0.9 nM). In contrast, the binding affinity of the dimer (KD = 1.6 nM) was approximately 2-fold lower than that of 1B2 Fab fragments. Both forms were able to inhibit recognition by the T cell, but the dimer was significantly more effective than the monomer. Inhibition was observed for T cell recognition of either the conventional ligand (a peptide bound to a class I product of the major histocompatibility complex) or the "superantigen" ligand (staphylococcal enterotoxin B bound to a class II product of the major histocompatibility complex). The results suggest that scFv dimers are the most active form of at least some scFv preparations and that the dimers may have the most potential for inhibiting detrimental in vivo T cell activity.
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana 61801.
authors.forename: B A
authors.initials: BA
authors.lastname: Schodin
authors.forename: D M
authors.initials: DM
authors.lastname: Kranz
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002999:Clone Cells; D003001:Cloning, Molecular; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D015202:Protein Engineering; D017510:Protein Folding; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; D016693:Receptors, Antigen, T-Cell, alpha-beta