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Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells.

Kuchař M, Vaňková L, Petroková H, Cerný J, Osička R, Pelák O, Sípová H, Schneider B, Homola J, Sebo P, Kalina T, Malý P - Proteins (2013)

Bottom Line: The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP-1 cells.Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL-23-driven expansion of IL-17-producing primary human CD4(+) T-cells.Thus, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti-inflammatory biologicals.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Ligand Engineering, Institute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech Republic.

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Immobilized REX ligands bind the soluble IL-23R-IgG receptor chimera. The REX-TolA-AVI variants REX009 and REX125, or ABDwt-TolA-AVI (ABDWT), were attached to the surface of SPR sensor over which the IL-23R-IgG receptor chimera was passed at 50 nM concentration. SA indicates sodium acetate buffer.
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fig07: Immobilized REX ligands bind the soluble IL-23R-IgG receptor chimera. The REX-TolA-AVI variants REX009 and REX125, or ABDwt-TolA-AVI (ABDWT), were attached to the surface of SPR sensor over which the IL-23R-IgG receptor chimera was passed at 50 nM concentration. SA indicates sodium acetate buffer.

Mentions: SPR biosensor binding analysis was used to further characterize the binding interactions between IL-23R and the REX ligands. The chip of a 4-channel SPR biosensor was functionalized with self-assembled monolayer of alkanethiols, to which His6-REX-TolA-AVI and His6-ABDwt-TolA-AVI proteins were attached. The response to IL-23R-IgG chimera interaction of such SPR biosensor is shown in Figure 7 for sensor surfaces functionalized with REX125, REX009 and control ABDwt proteins, respectively. It can be seen that the sensor response to IL-23R-IgG was much higher in the channels coated with the REX proteins than in the reference channel functionalized with ABDwt. Interestingly, the interaction of IL-23R-IgG with the REX variants was very sensitive to pH and salt composition of the running buffer. The complex was stable at pH 5.0 of the sodium acetate (SA) buffer, while it was very rapidly dissociated at pH 7.4 in PBS buffer. Similar results were obtained when IL-23R-IgG was immobilized to the sensor surface and REX proteins were flowed over the sensor (data not shown). The reference-compensated sensor responses to binding of IL-23R-IgG chimera to immobilized REX009, or REX125, respectively, at pH 5 were analyzed using an 1:1 interaction model in the kinetic BiaEvaluation software. Global fit of four concentrations in the range of 20–200nM indicated a dissociation constant Kd = 1.3 ± 0.7 × 10−9M for REX009. The binding affinity for REX125 could not be precisely determined due to a more complex interaction mode, but it was estimated to be in the order of 10−9M.


Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells.

Kuchař M, Vaňková L, Petroková H, Cerný J, Osička R, Pelák O, Sípová H, Schneider B, Homola J, Sebo P, Kalina T, Malý P - Proteins (2013)

Immobilized REX ligands bind the soluble IL-23R-IgG receptor chimera. The REX-TolA-AVI variants REX009 and REX125, or ABDwt-TolA-AVI (ABDWT), were attached to the surface of SPR sensor over which the IL-23R-IgG receptor chimera was passed at 50 nM concentration. SA indicates sodium acetate buffer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4285857&req=5

fig07: Immobilized REX ligands bind the soluble IL-23R-IgG receptor chimera. The REX-TolA-AVI variants REX009 and REX125, or ABDwt-TolA-AVI (ABDWT), were attached to the surface of SPR sensor over which the IL-23R-IgG receptor chimera was passed at 50 nM concentration. SA indicates sodium acetate buffer.
Mentions: SPR biosensor binding analysis was used to further characterize the binding interactions between IL-23R and the REX ligands. The chip of a 4-channel SPR biosensor was functionalized with self-assembled monolayer of alkanethiols, to which His6-REX-TolA-AVI and His6-ABDwt-TolA-AVI proteins were attached. The response to IL-23R-IgG chimera interaction of such SPR biosensor is shown in Figure 7 for sensor surfaces functionalized with REX125, REX009 and control ABDwt proteins, respectively. It can be seen that the sensor response to IL-23R-IgG was much higher in the channels coated with the REX proteins than in the reference channel functionalized with ABDwt. Interestingly, the interaction of IL-23R-IgG with the REX variants was very sensitive to pH and salt composition of the running buffer. The complex was stable at pH 5.0 of the sodium acetate (SA) buffer, while it was very rapidly dissociated at pH 7.4 in PBS buffer. Similar results were obtained when IL-23R-IgG was immobilized to the sensor surface and REX proteins were flowed over the sensor (data not shown). The reference-compensated sensor responses to binding of IL-23R-IgG chimera to immobilized REX009, or REX125, respectively, at pH 5 were analyzed using an 1:1 interaction model in the kinetic BiaEvaluation software. Global fit of four concentrations in the range of 20–200nM indicated a dissociation constant Kd = 1.3 ± 0.7 × 10−9M for REX009. The binding affinity for REX125 could not be precisely determined due to a more complex interaction mode, but it was estimated to be in the order of 10−9M.

Bottom Line: The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP-1 cells.Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL-23-driven expansion of IL-17-producing primary human CD4(+) T-cells.Thus, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti-inflammatory biologicals.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Ligand Engineering, Institute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech Republic.

Show MeSH
Related in: MedlinePlus