Binding affinity and inhibitory properties of a single-chain anti-T cell receptor antibody

Beth A. Schodin, David M. Kranz

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The antigen-specific function of T cells is mediated through a surface receptor (αβ). 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 α and β 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 ∼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.

Original languageEnglish (US)
Pages (from-to)25722-25727
Number of pages6
JournalJournal of Biological Chemistry
Issue number34
StatePublished - Dec 5 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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