Rational Design of Interleukin-21 Antagonist through Selective Elimination of the γC 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-21R␣) and the common ␥-chain (␥C), 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-21R␣, whereas the interaction between IL-21 and ␥C, albeit essential for signaling, is rather weak. The design of IL-21 analogues, which have lost most or all affinity toward the signaling ␥C 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 ␥C 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 ␥C affinity with undiminished IL-21R␣ affinity, were successfully identified. Functional studies confirmed that these two novel hIL-21 double mutants do act as hIL-21 antagonists. Interleukin (IL),2 -21 which is produced by activated CD4ϩ T cells, T-follicular helper cells, and natural killer T (NKT) cells, (1) has been demonstrated to exert pleiotropic effects on the proliferation, differentiation, and cytotoxicity of various classes of lymphoid cells. More recently, IL-21 has furthermore been shown to play a crucial role in the differentiation of CD4 ϩ T cells into T-helper 17 (TH 17 ) cells, a subset of T cells associated with development of inflammatory conditions and autoimmune diseases (2, 3).IL-21 signals through its receptor complex composed of the private chain IL-21R␣ and the common chain, ␥C, the latter of which is shared by five other cytokines: IL-2, IL-4, IL-7, IL-9, and IL-15 (1). Together these cytokines constitute the so-called ␥C family of cytokines. Despite the rather limited sequence homology (on average 15% sequence identity), these ␥C cytokines share a highly conserved overall four-helix bundle topology.Previously, we have reported the three-dimensional structure of human IL-21 (hIL-21) resolved by heteronuclear NMR spectroscopy (4). The structure of hIL-21 was shown to comprise a typical up-up-down-down four-␣-helical-bundle topology similar to that observed for several other type I cytokines, including the ␥C cytokines IL-2, IL-4, and IL-15. Noteworthy, a segment of hIL-21, including the helix C that by modeling is presumed to be important for IL-21R␣ binding, was demonstrated to represent a structurally unstable segment, not observed in the structures of other ␥C cytokines. A chimeric variant in which this flexible segment of hIL-21 had been exchanged with the corresponding IL-4 sequence was constructed and demonstrated to have a 10-fold increase in potency in a cell-based assay (4).Functional receptor ...

Insights into Cytokine–Receptor Interactions from Cytokine Engineering

“…Most cytokine engineering efforts to date have focused on receptor interface modulation with the goal of either enhancing affinity, as was implemented for IL-2 (80, 81), IL-4 (84), and IFN (85), or perturbing interaction, as was demonstrated for IL-2 (75, 86, 87), IL-4 (41), IFN (88), and IL-21 (77). Instances of cytokine engineering that have successfully redirected the specificity of a cytokine to alternative receptor subunits or narrowed the specificity of a cross-reactive cytokine (68, 78, 84) are less common.…”

Crystal Structure of Interleukin-21 Receptor (IL-21R) Bound to IL-21 Reveals That Sugar Chain Interacting with WSXWS Motif Is Integral Part of IL-21R

“…The extracellular domain (ECD) of IL-21R was expressed in HEK293 cells and purified as described (1).…”

“…IL-21 is a class I cytokine with a four-helix bundle structure arranged in an up-up-down-down topology typical for the class I cytokines (1). It exerts pleiotropic effects on both innate and adaptive immune responses.…”

Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies

“…The two arrows denote the residues mutated in helix D (Q116D and I119D) of the IL-21 mutein; these two mutations do not affect the ability of the mutein to bind IL-21R, but prevent signaling through the IL-21R/γ c complex, and are thought to be critical residues for γ c binding. 57,58 + T cells was added to BaF3/hIL-21R cells for 10 min and pSTAT3 measured to determine relative IL-21 concentration. An EC 90 dilution of conditioned media was mixed with a titration of IL-21 mAb clone 362.78 and incubated for 30 min prior to mixing with BaF3/hIL-21R cells.…”

“…7) that do not affect the mutein's ability to bind IL-21R, but do prevent signaling through the IL-21R/γ c complex. 57,58 Two of the mAbs that exhibited the best reactivity under denaturing conditions in western blot and peptide ELISA, mAbs 366.552 and 362.75, showed no reactivity to the human IL-21 mutein, and only mAb 362.75 was able to bind Peptide 4, which includes the amino acids affected in the IL-21 mutein ( Table 3). These data indicate that the region of IL-21 containing these two mutated amino acids (Q116D, I119D) comprises a critical feature in the epitope recognized by mAbs 366.552 and 362.75.…”