Kent Bondensgaard

The existence of multiple conformers of interleukin-21 directs engineering of a superpotent analogue.

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-up-down-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, α-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.

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

Background: The class I cytokine IL-21 exerts pleiotropic effects on innate and adaptive immunity. Results: We obtained the crystal structure of the partially glycosylated IL-21 receptor (IL-21R) bound to IL-21. Conclusion: A sugar chain is an integral part of IL-21R. Significance: This structure offers an insight into the putative role of the class I cytokine receptor signature motif. IL-21 is a class I cytokine that exerts pleiotropic effects on both innate and adaptive immune responses. It signals through a heterodimeric receptor complex consisting of the IL-21 receptor (IL-21R) and the common γ-chain. A hallmark of the class I cytokine receptors is the class I cytokine receptor signature motif (WSXWS). The exact role of this motif has not been determined yet; however, it has been implicated in diverse functions, including ligand binding, receptor internalization, proper folding, and export, as well as signal transduction. Furthermore, the WXXW motif is known to be a consensus sequence for C-mannosylation. Here, we present the crystal structure of IL-21 bound to IL-21R and reveal that the WSXWS motif of IL-21R is C-mannosylated at the first tryptophan. We furthermore demonstrate that a sugar chain bridges the two fibronectin domains that constitute the extracellular domain of IL-21R and anchors at the WSXWS motif through an extensive hydrogen bonding network, including mannosylation. The glycan thus transforms the V-shaped receptor into an A-frame. This finding offers a novel structural explanation of the role of the class I cytokine signature motif.

Comparison of the ability of wild type and stabilized human IgG4 to undergo Fab arm exchange with endogenous IgG4 in vitro and in vivo

Fab arm exchange by a stabilized anti-IL-31 IgG(4)S228P monoclonal antibody (mAb) was studied using physiologically relevant antibody concentrations and thiol exchange conditions, and directly compared to that of matched wild type IgG(4) (IgG(4)wt) and IgG(1) control antibodies. In vitro arm exchange between the test mAbs and a purified IgG(4)wt exchange partner was monitored using capillary isoelectric focusing and a size-exclusion peak shift assay. Arm exchange between the test mAbs and IgG exchange partners with unknown specificity was monitored using only the shift assay. Studies were performed using single isotype human and mouse mAbs, unfractionated human, mouse, and cynomolgus monkey IgG, and human serum as the sources of the exchange partners. In vitro studies using human serum demonstrated that anti-IL-31 IgG(4)S228P did not undergo significant Fab arm exchange with endogenous human IgG(4) whereas anti-IL-31 IgG(4)wt underwent rapid and extensive Fab arm exchange. The in vitro results were corroborated by in vivo studies in which mice were injected with a mixture of either form of the test mAb and an excess of non-specific human IgG(4) exchange partner.

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

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-21Rα) and the common γ-chain (γC), 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-21Rα, whereas the interaction between IL-21 and γC, albeit essential for signaling, is rather weak. The design of IL-21 analogues, which have lost most or all affinity toward the signaling γC 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 γC 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 γC affinity with undiminished IL-21Rα affinity, were successfully identified. Functional studies confirmed that these two novel hIL-21 double mutants do act as hIL-21 antagonists.

IL-21 Increased Potency Design

Publisher Summary This chapter focuses on cytokine variants which have distinguished themselves by exhibiting altered biological activity compared to the naturally occurring (wt) cytokine in terms of enhanced potency in biological assays. It also emphasizes the identification of super-agonists, which arise from the exchange of amino acid residues within the cytokine protein per se. The growth hormone (hGH) is a monomeric protein, and binds the hGH receptor in a 1:2 ratio via two non-identical sites, called sites 1 and 2. Variants of hGH exhibiting improved binding affinity to the hGH receptor have been identified through an extensive affinity maturation approach, and one such “super-binding” variant, hGH, encompassing a total of 15 single-substitutions, has been demonstrated to bind with a 400-fold enhanced affinity to the hGH receptor. A saturation mutagenesis employing a minimized yet fully active form of IL-3 has revealed that 1-2 percent out of a total of more than 700 individual single-site mutants exhibited enhanced activity, emphasizing that the naturally occurring cytokine represents but one active form of the protein molecule, albeit not the most active form. The cytokine IL-6 signals through a receptor complex composed of a selective chain, IL-6Rα, and a common chain, gp130, which is shared by other cytokine receptor complexes. The IL-6 receptor complex differs distinctly from the heterodimeric γC cytokine complexes in the sense that the gp130 chain by itself constitutes the signaling receptor subunit, while IL-6Rα solely provides affinity and even does so either in cis, when membrane-embedded along with gp130, or in trans, when present in solution or on another cell.