Design of Glycoengineered IL-4 Antagonists Employing Chemical and Biosynthetic Glycosylation

Interleukin-4 (IL-4) plays a key role in atopic diseases.It coordinatesT-helper cell differentiation to subtype 2, thereby directing defensetoward humoral immunity. Together with Interleukin-13, IL-4 furtherinduces immunoglobulin class switch to IgE. Antibodies of this typeactivate mast cells and basophilic and eosinophilic granulocytes,which release pro-inflammatory mediators accounting for the typicalsymptoms of atopic diseases. IL-4 and IL-13 are thus major targetsfor pharmaceutical intervention strategies to treat atopic diseases.Besides neutralizing antibodies against IL-4, IL-13, or its receptors,IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has beenevaluated in clinical trials for asthma treatment in the past; however,deficits such as short serum lifetime and potential immunogenicityamong others stopped further development. To overcome such deficits,PEGylation of therapeutically important proteins has been used toincrease the lifetime and proteolytic stability. As an alternative,glycoengineering is an emerging strategy used to improve pharmacokineticsof protein therapeutics. In this study, we have established differentstrategies to attach glycan moieties to defined positions in IL-4.Different chemical attachment strategies employing thiol chemistrywere used to attach a glucose molecule at amino acid position 121,thereby converting IL-4 into a highly effective antagonist. To enhancethe proteolytic stability of this IL-4 antagonist, additional glycanstructures were introduced by glycoengineering utilizing eucaryoticexpression. IL-4 antagonists with a combination of chemical and biosyntheticglycoengineering could be useful as therapeutic alternatives to IL-4neutralizing antibodies already used to treat atopic diseases.