Preparation and characterization of a disulfide-stabilized Fv fragment of the anti-Tac antibody: comparison with its single-chain analog.
Recombinant DNA techniques now allow the production of "mini-antibodies" called Fv fragments. These have been produced either as the independent variable domains of the heavy and light chains non-covalently associated in one-to-one stoichiometry or as single-chain gene products with the two domains linked by an intervening peptide sequence. Although Fv fragments can have excellent binding properties, they are often difficult to produce in good yield and lack the characteristic stability of whole antibodies. To improve the stability of the Fv molecule, we have introduced a cysteine residue into conserved framework regions of both the heavy and light variable domains from the anti-Tac antibody at positions compatible with the formation of an interdomain disulfide linkage (i.e. VH-44 and VL-99). The mutant subunits form a disulfide-bonded Fv molecule, which binds to the alpha-subunit of the IL2 receptor (IL2R alpha) with an affinity identical to that of humanized anti-Tac IgG. This disulfide-stabilized Fv (dsFv) proved to be substantially more resistant to denaturation by heat or urea treatment than the single-chain Fv (scFv). Furthermore, the yield of dsFv is -four-fold higher than that of the single-chain analog.
Webber
K O
KO
Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Reiter
Y
Y
Brinkmann
U
U
Kreitman
R
R
Pastan
I
I
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001483:Base Sequence; D001667:Binding, Competitive; D017076:Computer-Aided Design; D017931:DNA Primers / Q000737:chemistry; D004220:Disulfides; D004926:Escherichia coli; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015375:Receptors, Interleukin-2 / Q000276:immunology*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D017931:DNA Primers; D004220:Disulfides; D007128:Immunoglobulin Fragments; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; C055966:immunoglobulin Fv