STRUCTURE-FUNCTION STUDIES OF INTERLEUKIN-15 USING SITE-SPECIFIC MUTAGENESIS, POLYETHYLENE-GLYCOL CONJUGATION, AND HOMOLOGY MODELING

Interleukin (IL)-15 is a multifunctional cytokine that shares many bio logical activities with IL-2. This functional overlap, as well as rece ptor binding subunits shared by IL-15 and IL-2, suggests tertiary stru ctural similarities between these two cytokines. In this study, recomb inant human IL-15 was PEGylated via lysine-specific conjugation chemis try in order to extend the circulation half-life of this cytokine. Alt hough PEGylation did extend the beta-elimination circulation half-life of IL-15 by greater than 50-fold, the biological activity of polyethy lene glycol (PEG)-IL-15 was significantly altered. Specifically, PEG-I L-15 lost its ability to stimulate the proliferation of CTLL but took on the properties of a specific IL-15 antagonist in vitro. In comparin g sequence alignments and molecular models for IL-2 and IL-15, it was noted that lysine residues resided in regions of IL-15 that may have s electively disrupted receptor subunit binding. We hypothesized that PE Gylation of IL-15 interferes with beta but not alpha receptor subunit binding, resulting in the IL-15 antagonist activity observed in vitro. The validity of this hypothesis was tested by engineering site-specif ic mutants of human IL-15 as suggested by the IL-15 model (IL-15D8S an d IL-15Q108S block beta and gamma receptor subunit binding, respective ly). As with PEG-IL-15, these mutants were unable to stimulate CTLL pr oliferation but were able to specifically inhibit the proliferation of CTLL in response to unmodified IL-15. These results supported our mod el of IL-15 and confirmed that interference of beta receptor subunit b inding by adjacent PEGylation could be responsible for the altered bio logical activity observed for PEG-IL-IB.