Effect of VH and VL consensus sequence-specific primers on the binding and neutralizing potential of a single-chain FV directed towards HuIFN-gamma.
We have previously reported on the cloning and bacterial expression of a biologically active scFv antibody fragment (scFv-D9D10) derived from the mouse anti-human interferon-gamma (HuIFN-gamma) antibody, D9D10. Since the variable (V) regions were isolated by means of VH and VL consensus sequence-specific PCR primers and cloned in an expression vector relying on primer-incorporated restriction sites, some amino acids (aa) at the N- and C-terminal ends of the cloned V domains were expected to differ from the corresponding ones in the natural D9D10 antibody. Therefore, the naturally occurring sequences of both V domains were isolated by means of traditional cDNA synthesis procedures. In comparison with scFv-D9D10, the "natural" V sequences differed in three aa in VH and three in VL. The V domains of scFv-D9D10 were adapted to their natural sequence by means of PCR-directed mutagenesis to yield scFv-D9D10N. Comparison of the binding and neutralizing potentials of both antibody fragments did not reveal differences in either of both activities. In addition, their affinities for HuIFN-gamma were found to be equal. These results show that murine VH and VL consensus-specific primers can yield antibody fragments having functional properties equivalent to those of the natural scFv. Information on the impact of the use of V-specific primers on kinetics of interaction between the recombinant antibody and the corresponding antigen is important for the development of most engineered antibodies or their fragments.
Froyen
G
G
Rega Institute for Medical Research, University of Leuven, Belgium.
Hendrix
D
D
Ronsse
I
I
Fiten
P
P
Martens
E
E
Billiau
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D007371:Interferon-gamma