FIGURE 4.

Characterization of antagonistic IL-21 variants. A, biological activity of IL-21 variants compared with wt IL-21 in a NK92 cell proliferation assay. B, inhibition of wt IL-21 induced NK92 cell proliferation by antagonistic IL-21 variants; NK92 cells were incubated with wt IL-21 at the IL-21 concentration inducing 50% of maximal response, followed by adding serial dilutions of antagonistic IL-21 variants or PBS as blank control. After 72 h of incubation, proliferation was quantified by Alamar-Blue reduction. C, relative affinities of antagonistic IL-21 variants toward IL-21Rα were measured using an ALPHAscreen competition test. Increasing concentrations of IL-21 variants were added to compete the binding of biotinylated IL-21 to IL-21Rα-His₆. The curves represent one experiment performed in triplicate, and the error bars represent the S.D. All of the experiments were repeated at least three times with similar results. IL-21 variants were expressed by HEK293 cells and used as crude supernatants. Protein concentrations were measured relative to wt IL-21 using an ALPHAscreen-based assay.