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Development of interleukin-1 receptor antagonist mutants with enhanced antagonistic activity in vitro and improved therapeutic efficacy in collagen-induced arthritis.

Dahlén, Eva LU ; Barchan, Karin; Herrlander, Daniel; Höjman, Patrik; Karlsson, Marie LU ; Ljung, Lill; Andersson, Mats; Bäckman, Eva; Malmborg Hager, Ann-Christin LU and Walse, Björn, et al. (2008) In Journal of Immunotoxicology 5(2). p.189-199
Abstract
Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring inhibitor of the pro-inflammatory interleukin-1-mediated activation of the interleukin-1 receptor (IL-1R). Although wild-type IL-1Ra is used for treatment of inflammatory diseases, its effect is moderate and/or short-lived. The objective of this study was to generate IL-1Ra mutants with enhanced antagonistic activity for potential therapeutic use. Using a directed evolution approach in which libraries of IL-1Ra gene mutants were generated and screened in functional assays, mutants with desired properties were identified. Initially, diversity was introduced into the IL-1Ra using random mutagenesis. Mutations resulting in enhanced antagonistic activity were identified by... (More)
Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring inhibitor of the pro-inflammatory interleukin-1-mediated activation of the interleukin-1 receptor (IL-1R). Although wild-type IL-1Ra is used for treatment of inflammatory diseases, its effect is moderate and/or short-lived. The objective of this study was to generate IL-1Ra mutants with enhanced antagonistic activity for potential therapeutic use. Using a directed evolution approach in which libraries of IL-1Ra gene mutants were generated and screened in functional assays, mutants with desired properties were identified. Initially, diversity was introduced into the IL-1Ra using random mutagenesis. Mutations resulting in enhanced antagonistic activity were identified by screening in a reporter cell assay. To further enhance the antagonistic activity, selected mutations were recombined using the DNA recombination technology Fragment-INduced Diversity (FIND). Following three rounds of FIND recombination, several mutants with up to nine times enhanced antagonistic activity (mean IC50 +/- SEM value: 0.78 +/- 0.050 vs. 6.8 +/- 1.1 ng/ml for mutant and wild-type, respectively) were identified. Sequence analysis identified the mutations D47N, E52R and E90Y as being most important for this effect, however, the mutations P38Y, H54R, Q129L and M136N further enhanced the antagonistic function. Analysis of identified mutations in protein models based on the crystal structure of the IL-1Ra/IL-1R complex suggested that mutations found to enhance the antagonistic activity had a stabilizing effect on the IL-1Ra mutants or increased the affinity for the IL-1R. Finally, the therapeutic effect of one mutant was compared to that of wild-type IL-1Ra in collagen-induced arthritis in mice. Indeed, the enhanced antagonistic effect of the mutants observed in vitro was also seen in vivo. In conclusion, these results demonstrate that directed evolution of IL-1Ra is an effective means of generating highly potent therapeutic proteins. (Less)
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Contribution to journal
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published
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Journal of Immunotoxicology
volume
5
issue
2
pages
189 - 199
publisher
Informa Healthcare
external identifiers
  • pmid:18569390
  • scopus:45849135653
ISSN
1547-6901
DOI
10.1080/15476910802131477
language
English
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yes
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0be7bb61-ee8e-4ec9-ac89-a29ed8d427f8 (old id 1168603)
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http://www.ncbi.nlm.nih.gov/pubmed/18569390?dopt=Abstract
date added to LUP
2008-07-02 14:01:05
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2017-01-01 07:30:42
@article{0be7bb61-ee8e-4ec9-ac89-a29ed8d427f8,
  abstract     = {Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring inhibitor of the pro-inflammatory interleukin-1-mediated activation of the interleukin-1 receptor (IL-1R). Although wild-type IL-1Ra is used for treatment of inflammatory diseases, its effect is moderate and/or short-lived. The objective of this study was to generate IL-1Ra mutants with enhanced antagonistic activity for potential therapeutic use. Using a directed evolution approach in which libraries of IL-1Ra gene mutants were generated and screened in functional assays, mutants with desired properties were identified. Initially, diversity was introduced into the IL-1Ra using random mutagenesis. Mutations resulting in enhanced antagonistic activity were identified by screening in a reporter cell assay. To further enhance the antagonistic activity, selected mutations were recombined using the DNA recombination technology Fragment-INduced Diversity (FIND). Following three rounds of FIND recombination, several mutants with up to nine times enhanced antagonistic activity (mean IC50 +/- SEM value: 0.78 +/- 0.050 vs. 6.8 +/- 1.1 ng/ml for mutant and wild-type, respectively) were identified. Sequence analysis identified the mutations D47N, E52R and E90Y as being most important for this effect, however, the mutations P38Y, H54R, Q129L and M136N further enhanced the antagonistic function. Analysis of identified mutations in protein models based on the crystal structure of the IL-1Ra/IL-1R complex suggested that mutations found to enhance the antagonistic activity had a stabilizing effect on the IL-1Ra mutants or increased the affinity for the IL-1R. Finally, the therapeutic effect of one mutant was compared to that of wild-type IL-1Ra in collagen-induced arthritis in mice. Indeed, the enhanced antagonistic effect of the mutants observed in vitro was also seen in vivo. In conclusion, these results demonstrate that directed evolution of IL-1Ra is an effective means of generating highly potent therapeutic proteins.},
  author       = {Dahlén, Eva and Barchan, Karin and Herrlander, Daniel and Höjman, Patrik and Karlsson, Marie and Ljung, Lill and Andersson, Mats and Bäckman, Eva and Malmborg Hager, Ann-Christin and Walse, Björn and Joosten, Leo and van den Berg, Wim},
  issn         = {1547-6901},
  language     = {eng},
  number       = {2},
  pages        = {189--199},
  publisher    = {Informa Healthcare},
  series       = {Journal of Immunotoxicology},
  title        = {Development of interleukin-1 receptor antagonist mutants with enhanced antagonistic activity in vitro and improved therapeutic efficacy in collagen-induced arthritis.},
  url          = {http://dx.doi.org/10.1080/15476910802131477},
  volume       = {5},
  year         = {2008},
}