The Existence of Multiple Conformers of Interleukin-21 Directs Engineering of a Superpotent Analogue

Abstract

The high resolution three-dimensional structure of human interleukin (hIL)-21 has been resolved by heteronuclear NMR spectroscopy. Overall, the hIL-21 structure is dominated by a well defined central four-helical bundle, arranged in an up-updown-down topology, as observed for other cytokines. A segment of the hIL-21 molecule that includes the third helical segment, helix C, is observed to exist in two distinct and interchangeable states. In one conformer, the helix C segment is presented in a regular, ␣-helical conformation, whereas in the other conformer, this segment is largely disordered. A structure-based sequence alignment of hIL-21 with receptor complexes of the related cytokines, interleukin-2 and -4, implied that this particular segment is involved in receptor binding. An hIL-21 analog was designed to stabilize the region around helix C through the introduction of a segment grafted from hIL-4. This novel hIL-21 analog was demonstrated to exhibit a 10-fold increase in potency in a cellular assay. Interleukin (IL)2 -21 is a recently identified type 1 cytokine, which is secreted as a 133-amino acid protein by activated CD4 ϩ T cells (1). The IL-21 cytokine has been demonstrated to possess potent stimulatory effects on the proliferation, differentiation, and activation of several classes of hematopoietic cells, including B-cells, T-cells, and NK-cells. The biological effects of IL-21 are mediated via activation of the IL-21 receptor complex, which is composed of an IL-21 private receptor chain (IL-21R␣) in complex with the common ␥ chain (␥ c ), which similarly constitutes an essential component of the signaling receptor complex of the cytokines IL-2, IL-4, IL-7, IL-9, and IL-15. These cytokines thus constitute a subfamily referred to as common ␥ chain cytokines, with IL-21 being the most recently added member (2).Within the common ␥ chain family of cytokines, high resolution structural information has been obtained through x-ray crystallography and NMR spectroscopy for IL-2 and IL-4 (3-8). It is apparent from these studies that IL-2 and IL-4 along with other type 1 cytokines, including IL-6 and granulocyte-macrophage colony-stimulating factor, share a common overall topology in their structures despite a distant homology in sequence (9). The common structural motif of these proteins consists of a central four-helical bundle, arranged in an up-updown-down topology, connected by loops that are characterized by a high degree of structural freedom, a considerable difference in loop length, and variation in the number and positioning of stabilizing disulfide bridges.Crystal structures have also been reported for IL-2 and IL-4 in complex with the corresponding private chains and, in the case of IL-2, the common ␥ chain (10 -12). IL-2 is distinct from both IL-4 and IL-21 by having two private receptor chains, IL-2R␣ and IL-2R␤, where IL-2R␤ is homologous to IL-4R␣ and IL-21R␣. Only minor structural differences are observed between the free and receptor-bound forms of IL-2 and IL-4, indicating that only slight stru...

In vitro potency, pharmacokinetic profiles and pharmacological activity of optimized anti-IL-21R antibodies in a mouse model of lupus

“…This trend toward basic character could reflect interaction with the proposed IL-21 binding interface of IL-21R, which is believed to have a high negative charge. 24,25 Different sequence classes appeared to provide optimal binding to mouse and human receptors, e.g., in binding assays in which scFvs competed with the 18A5 scFv for binding to mIL-21R (data not shown). Clones with isoleucine or methionine at the first position in the V L CDR3 were among the strongest binders to mIL-21R, but bound more weakly than most to hIL-21R.…”

Rational Design of Interleukin-21 Antagonist through Selective Elimination of the γC Binding Epitope

“…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).…”

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.…”