Rational design of interleukin-21 antagonist through selective elimination of the gammaC binding epitope

Abstract

The cytokine interleukin (IL)-21 exerts pleiotropic effects acting through innate as well as adaptive immune responses. The activities of IL-21 are mediated through binding to its cognate receptor complex composed of the IL-21 receptor private chain (IL-21Ralpha) and the common gamma-chain (gammaC), the latter being shared by IL-2, IL-4, IL-7, IL-9, and IL-15. The binding energy of the IL-21 ternary complex is predominantly provided by the high affinity interaction between IL-21 and IL-21Ralpha, whereas the interaction between IL-21 and gammaC, albeit essential for signaling, is rather weak. The design of IL-21 analogues, which have lost most or all affinity toward the signaling gammaC chain, while simultaneously maintaining a tight interaction with the private chain, would in theory represent candidates for IL-21 antagonists. We predicted the IL-21 residues, which compose the gammaC binding epitope using homology modeling and alignment with the related cytokines, IL-2 and IL-4. Next we systematically analyzed the predicted binding epitope by a mutagenesis study. Indeed two mutants, which have significantly impaired gammaC affinity with undiminished IL-21Ralpha affinity, were successfully identified. Functional studies confirmed that these two novel hIL-21 double mutants do act as hIL-21 antagonists.

FIGURE 1.

A, sequence alignment of hIL-21, hIL-2, and hIL-4 based on the structural alignment and adjusted by hand. γC binding residues on IL-21 predicted from alignment with IL-2 and IL-4 receptor complexes are boxed in the alignment together with the corresponding residues in IL-2 and IL-4. Three additional γC binding residues identified from the model (M7, E100, E109) are only boxed in the sequence of hIL-21. The numbering follows hIL-21. B, homology model of the IL-21/IL-21Rα/γC complex generated by the Modeler program. The structures of IL-21, IL-21Rα, and γC are depicted as blue, green, and red ribbons, respectively. C, potential γC binding residues of hIL-21 are depicted as yellow sticks.

FIGURE 2.

Binding interface of IL-21Rα depicted on IL-21. IL-21 residues mutated to aspartic acid or residues containing opposite charge are labeled on the structure and color-coded according to effect on IL-21Rα and γC binding affinity. Residues shown in red and green have ≥2-fold impaired binding affinity toward either IL-21Rα or γC, respectively, whereas residues having ≥2-fold impaired IL-21Rα and γC binding affinities are shown in yellow (See Tables 1 and 2).

FIGURE 3.

Biological activity of IL-21 variants in a NK92 cell proliferation assay. A, variants with aspartate mutations in positions 116, 120, and 123 compared with wt IL-21. B, screen of position 116 variants compared with wt IL-21. In each case, NK92 cells were incubated with serial 10-fold dilutions of wt IL-21 or variants for 72 h, and thereafter proliferation was quantified by Alamar-Blue reduction. The curves represent one experiment performed in triplicate, and the error bars represent the S.D. Experiments were repeated at least three times with similar results. Activity is expressed as a percentage of maximal response. 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.

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.