isrctn.org Identifier: ISRCTN54371254.

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Interleukin‐4 (IL‐4) represents a prototypic lymphokine (for a recent review see Paul, 1991). It promotes differentiation of B‐cells and the proliferation of T‐ and B‐cell, and other cell types of the lymphoid system. An antagonist of human IL‐4 was discovered during the studies presented here after Tyr124 of the recombinant protein had been substituted by an aspartic acid residue. This IL‐4 variant, Y124D, bound with high affinity to the IL‐4 receptor (KD = 310 pM), but retained no detectable proliferative activity for T‐cells and inhibited IL‐4‐dependent T‐cell proliferation competitively (K(i) = 620 pM). The loss of efficacy in variant Y124D was estimated to be greater than 100‐fold on the basis of a weak partial agonist activity for the very sensitive induction of CD23 positive B‐cells. The substitution of Tyr124 by either phenylalanine, histidine, asparagine, lysine or glycine resulted in partial agonist variants with unaltered receptor binding affinity and relatively small deficiencies in efficacy. These results demonstrate that high affinity binding and signal generation can be uncoupled efficiently in a ligand of a receptor belonging to the recently identified hematopoietin receptor family. In addition we show for the first time, that a powerful antagonist acting on the IL‐4 receptor system can be derived from the IL‐4 protein.

Human interleukin-4 possesses two distinct sites for receptor activation. A signalling site, comprising residues near the C-terminus on helix D, determines the efficacy of interleukin-4 signal transduction without affecting the binding to the interleukin-4 receptor a subunit. A complete antagonist and a series of low-efficacy agonist variants of human interleukin-4 could be generated by introducing combinations of two or three negatively charged aspartic acid residues in this site at positions 121, 124, and 125. One of the double variants, designated [R121D,Y124D]interleukin-4, with replacements of both Argl21 and Tyrl24 by aspartic acid residues was completely inactive in all analysed cellular responses. The loss of efficacy in [R121D,Y 124Dlinterleukin-4 is estimated to be larger than 2000-fold. Variant [R121D,Y124D]interleukin-4 was also a perfect antagonist for inhibition of interleukin-13-dependent responses in B-cells and the TF-1 cell line with a K, value of approximately 100 pM. In addition, inhibition of both interleukin-4-induced and interleulun-13-induced responses could be obtained by monoclonal antibody X2/45 raised against interleukin-4Re,, the extracellular domain of the interleukin-4 receptor a subunit. These results indicate that efficient interleukin-4 antagonists can be designed on the basis of a sequential two-step activation model. In addition, the experiments indicate the functional participation of the interleukin-4 receptor a subunit in the interleukin-13 receptor system.Human interleukin-4 (IL-4) activates its cognate receptor system by sequential binding to two different receptor subunits. The first high-affinity binding step (& 100 pM) involves the interaction between IL-4 receptor a subunit (Beckmann et al., 1992;Galizzi et al., 1990;Idzerda et al., 1990) and IL-4 amino acid residues located on helices A and C (Kruse et al., 1993;Ramanathan et al., 1993). The subsequent second binding step involves a signalling site on helix D of IL-4 (Kruse et al., 1992) and a further receptor subunit, which according to recent evidence (Kondo et al., 1993;Russell et al., 1993) is probably the y chain of the IL-2 receptor system, now designated yc.Such a sequential two-step binding mechanism leading to receptor activation suggests a rationale for the design of antagonistic cytokine variants (Cunningham et al., 1991 ;De Vos et al., 1992). If the first high-affinity binding site is retained and the second signalling site is either destroyed by (Fuh et al., 1992;Kruse et al., 1992) or blocked by monoclonal antibodies (Reusch et al., 1994), the oligomerisation process stops at the level of the unproductive 1 : 1 complex between the cytokine and the first receptor subunit. The signalling site of human IL-4 comprises side chains of Argl21, Tyrl24 and Ser125. Previous results have shown that variants [R121D]IL-4 and [S225D]IL-4 have approximately 30% partial agonist activity and variant [Y124D]IL-4 has less than 1 % partial agonist activity for T-cell proliferation compared to wild-type IL-4. High-affinit...

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