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Nishida, Y. et al.

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.[1]

References

Cloning and expression of a single-chain Fv fragment specific for the human interleukin 18 receptor. Nishida, Y., Torigoe, K., Aizawa, Y., Okura, T., Mori, T., Yamauchi, H., Tanimoto, T., Ikeda, M., Ikegami, H., Kurimoto, M. Hybridoma (1998) [Pubmed]

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