ABSTRACT
IL-4 signaling into a cell occurs via assembly of a receptor complex that consists of a high-affinity IL-4Rα chain and a low affinity chain, where the low-affinity chain is either γc or IL-13Rα1. It has been previously shown that mutational disruption of the low affinity interface in the IL-4DM (double mutein) yields an antagonist that inhibits IL-4 as well as IL-13-dependent responses. The present study reveals that new types of IL-4 antagonists can be generated by site-specific chemical modification. The chemically modified IL-4 analogues consist of (1) mixed disulfides created by refolding IL-4 cysteine muteins in the presence of different thiol compounds or (2) maleimide conjugates created by modifying cysteine muteins with maleimide derivatives. IL-4 analogues chemically modified at position 121 retain marginal binding affinity to γc or IL-13Rα1 receptor ectodomains during SPR interaction analysis. The biological activity of the analogues is strongly reduced in HEK-Blue IL-4/IL-13 cells as well as in Jurkat cells. Since the IL-4 analogues modified at position 121 have the ability to inhibit γc (IL-4)- and IL13Rα1 (IL-4/IL-13)-dependent responses in Jurkat and HEK-Blue cell lines, they effectively act as IL-4 antagonists. The results of our IL-4 study provide the first example of a cytokine that is transformed into a competitive inhibitor by site-specific chemical modification.
Subject(s)
Interleukin-13/antagonists & inhibitors , Interleukin-4/analogs & derivatives , Interleukin-4/antagonists & inhibitors , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Interleukin-4/chemistry , Jurkat Cells , Models, Molecular , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
Interleukin-4 (IL-4) is a prototypical regulator protein of the immune system that is crucial for the pathogenesis and maintenance of asthma and other atopic diseases. It, together with IL-13, uses the IL-4 receptor α chain (IL-4Rα) to signal into immune and other cells. An IL-4 mutein acting as a dual IL-4/IL-13 receptor antagonist is in clinical development. Here, it is described how IL-4 muteins containing a single engineered cysteine with a free thiol can be prepared and used for site-specific chemical modification. The muteins were initially expressed in E. coli, refolded, and purified, but not in a fully reduced nonconjugated form. Attempts to reduce the cysteine chemically failed because the native disulfide bonds of IL-4 were also reduced under similar conditions. Therefore, an enzymatic procedure was developed to reduce glutathionylated IL-4 cysteine muteins employing glutaredoxin and reduced glutathione. Cysteine muteins engineered at four different positions around the IL-4Rα binding site were enzymatically reduced at different rates. All muteins were prepared with free thiol in reasonable yield and were modified by N-ethylmaleimide (NEM) or maleimido-PEG. The effect on IL-4Rα binding of cysteine substitution and of the site-specific modification by glutathione, N-ethylmaleimide (NEM), or a branched 2.36 kDa poly(ethylene glycol) (PEG) will be discussed.