Cloning and expression of a single-chain Fv fragment specific for the human interleukin 18 receptor.
We have recently reported the successful purification and characterization of the human interleukin 18 (IL-18) receptor (R) using a monoclonal antibody (MAb) of the IgM kappa class (117-10C) after immunizing mice with cells of the Hodgkin's disease-derived cell line L428. This antibody is specific for the human IL-18R and inhibits the bioactivity of IL-18. Here we report on the polymerase chain reaction (PCR)-assisted cloning and sequencing of cDNAs encoding the variable regions of the light and heavy chains of 117-10C. We expressed the antibody in the form of a single-chain Fv fragment (scFv) in Chinese Hamster Ovary (CHO) cells and purified it from culture supernatants by chromatography. The purified scFv has an affinity for IL-18R of 5.6 x 10(8) M(-1), whereas 117-10C binds to the receptor with an affinity constant of 3.6 x 10(9) M(-1). Since 117-10C is of the IgM class, it is expected to be in the pentamer form and should theoretically therefore bind IL-18R with 10 times the affinity of the single-chain fragment may explain the difference in the affinity constants for the two molecules. The inhibitory efficiency of 117-10C was found to be 6-fold that of scFv in an interferon gamma (IFN-gamma)-inducing assay on the IL-18-responsive cell line KG-1. In conclusion, we have produced a single-chain fragment of a murine anti-human IL-18R antibody that is as potent at binding IL-18 as the parent antibody, and may be useful in neutralizing the cytokine in human conditions associated with high production of IL-18.
Nishida
Y
Y
Fujisaki Institute, Hayashibara Biochemical Laboratories, Inc., Okayama, Japan.
Torigoe
K
K
Aizawa
Y
Y
Okura
T
T
Mori
T
T
Yamauchi
H
H
Tanimoto
T
T
Ikeda
M
M
Ikegami
H
H
Kurimoto
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D007135:Immunoglobulin Variable Region; D053727:Interleukin-18 Receptor alpha Subunit; D051379:Mice; D008969:Molecular Sequence Data; D018123:Receptors, Interleukin / Q000235:genetics* / Q000276:immunology; D053724:Receptors, Interleukin-18
D000911:Antibodies, Monoclonal; C508572:IL18R1 protein, human; C508573:Il18r1 protein, mouse; D007128:Immunoglobulin Fragments; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D053727:Interleukin-18 Receptor alpha Subunit; D018123:Receptors, Interleukin; D053724:Receptors, Interleukin-18; C055966:immunoglobulin Fv