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 biological activities with IL-2. This functional overlap, as well as receptor binding subunits shared by IL-15 and IL-2, suggests tertiary structural similarities between these two cytokines. In this study, recombinant human IL-15 was PEGylated via lysine-specific conjugation chemistry in order to extend the circulation half-life of this cytokine. Although PEGylation did extend the beta-elimination circulation half-life of IL-15 by greater than 50-fold, the biological activity of polyethylene glycol (PEG)-IL-15 was significantly altered. Specifically, PEG-IL-15 lost its ability to stimulate the proliferation of CTLL but took on the properties of a specific IL-15 antagonist in vitro. In comparing 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 selectively disrupted receptor subunit binding. We hypothesized that PEGylation 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-specific mutants of human IL-15 as suggested by the IL-15 model (IL-15D8S and IL-15Q108S block beta and gamma receptor subunit binding, respectively). As with PEG-IL-15, these mutants were unable to stimulate CTLL proliferation but were able to specifically inhibit the proliferation of CTLL in response to unmodified IL-15. These results supported our model of IL-15 and confirmed that interference of beta receptor subunit binding by adjacent PEGylation could be responsible for the altered biological activity observed for PEG-IL-15.
Pettit
D K
DK
Department of Analytical Chemistry and Formulation, Immunex Corporation, Seattle, Washington 98101, USA. pettitd@immunex.com
Bonnert
T P
TP
Eisenman
J
J
Srinivasan
S
S
Paxton
R
R
Beers
C
C
Lynch
D
D
Miller
B
B
Yost
J
J
Grabstein
K H
KH
Gombotz
W R
WR
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019409:Interleukin-15 / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D007376:Interleukin-2 / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011092:Polyethylene Glycols / Q000378:metabolism*; D011487:Protein Conformation; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship
D019409:Interleukin-15; D007376:Interleukin-2; D011092:Polyethylene Glycols