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. 2009 Sep 15;183(6):3598-607.
doi: 10.4049/jimmunol.0901244. Epub 2009 Aug 26.

Novel human interleukin-15 agonists

Xiaoyun Zhu  1 , Warren D Marcus, Wenxin Xu, Hyung-il Lee, Kaiping Han, Jack O Egan, Jason L Yovandich, Peter R Rhode, Hing C Wong
Affiliations

VSports在线直播 - Novel human interleukin-15 agonists

Xiaoyun Zhu et al. J Immunol. .

Abstract

IL-15 is an immunostimulatory cytokine trans-presented with the IL-15 receptor alpha-chain to the shared IL-2/IL-15Rbeta and common gamma-chains displayed on the surface of T cells and NK cells. To further define the functionally important regions of this cytokine, activity and binding studies were conducted on human IL-15 muteins generated by site-directed mutagenesis. Amino acid substitutions of the asparagine residue at position 72, which is located at the end of helix C, were found to provide both partial agonist and superagonist activity, with various nonconservative substitutions providing enhanced activity. Particularly, the N72D substitution provided a 4-5-fold increase in biological activity of the IL-15 mutein compared with the native molecule based on proliferation assays with cells bearing human IL-15Rbeta and common gamma-chains. The IL-15N72D mutein exhibited superagonist activity through improved binding ability to the human IL-15Rbeta-chain. However, the enhanced potency of IL-15N72D was not observed with cells expressing the mouse IL-15Ralpha-IL-15Rbeta-gamma(c) complex, suggesting that this effect is specific to the human IL-15 receptor. The enhanced biological activity of IL-15N72D was associated with more intense phosphorylation of Jak1 and Stat5 and better anti-apoptotic activity compared with the wild-type IL-15 VSports手机版. IL-15N72D superagonist activity was also preserved when linked to a single-chain TCR domain to generate a tumor-specific fusion protein. Thus, the human IL-15 superagonist muteins and fusions may create opportunities to construct more efficacious immunotherapeutic agents with clinical utility. .

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Figures (V体育2025版)

FIGURE 1
FIGURE 1
Characterization of c264scTCR/hIL-15 fusion proteins. A, Schematic representation of the fusion protein of c264scTCR/hIL-15 or c264scTCR/hIL-15N72D. B, CTLL-2 cells were incubated with or without c264scTCR/birA or c264scTCR/hIL-15 initially and then detected with either biotinylated anti-human TCR Cβ (BF1) and PE-conjugated streptavidin (left) or PE-conjugated p53 (aa264-272)/HLA-A2 tetramer (right). C, 32Dβ cells were incubated with increasing concentrations of c264scTCR/hIL-15 (□) or recombinant hIL-15 (■) for 48 h prior to addition of WST-1 for 4 h and cell proliferation was quantitated by absorbance reading at 440 nm to assess formazan levels. The data points shown are means (±SEM) of triplicate samples and the lines represent sigmoidal dose-response curve fit for EC50 determination. The results are representative of at least three experiments.
FIGURE 2
FIGURE 2
Effect of wild-type hIL-15 and muteins on 32Dβ cell proliferation. 32Dβ cells (hIL-15Rβ-mγ-positive) were incubated with increasing concentrations of hIL-15-containing proteins for 48 h and proliferation assays were performed as described in Figure 1. A, Dose-responses curves of 32Dβ cell proliferation assessed with different concentrations of c264scTCR/hIL-15 wild type (■) and c264scTCR/hIL-15N72D (□) fusions. B, Proliferation assays of 32Dβ cells using c264scTCR/hIL-15 and nine hIL-15 mutein fusion proteins were carried out. The activity based on EC50 of c264scTCR/hIL15 wild-type fusion was normalized to 100% and the relative activities of mutein fusions were determined. C, Dose-responses curves of 32Dβ cell proliferation assessed with different concentrations of rhIL-15 wild type (■) and rhIL-15N72D (□) proteins. The results are representative of at least three independent experiments.
FIGURE 3
FIGURE 3
Effect of hIL-15 wild type and N72D mutein proteins on CTLL-2 cell proliferation. CTLL-2 cells (mIL-15Rα-mIL-15Rβ-mγc-positive) were incubated with increasing concentrations of hIL-15-containing proteins for 48 h and proliferation assays were performed as described in Figure 1. A, Dose-responses curves of CTLL-2 cell proliferation assessed with different concentrations of c264scTCR/hIL-15 wild type (■) and c264scTCR/hIL-15N72D (□) fusions. B, Dose-responses curves of CTLL-2 cell proliferation assessed with different concentrations of rhIL-15 wild type (■) and rhIL-15N72D (□) proteins. The results are representative of at least three experiments.
FIGURE 4
FIGURE 4
Effect of hIL-15 wild type and N72D mutein proteins on TF-1β cell proliferation. A, TF-1 cells and TF-1β cells were stained with anti-CD122 (black thick line) or an isotype control (gray shade) to verify expression of the hIL-15Rβ gene. B, TF-1β cells (hIL-15Rα-hIL-15Rβ-hγc-positive) were incubated with increasing concentrations of rhIL-15 wild type (■) and rhIL-15N72D (□) for 48 h and proliferation assays were performed as described in Figure 1. The results are representative of at least three experiments.
FIGURE 5
FIGURE 5
hIL-15N72D exhibits higher binding affinity than wild type hIL-15 to hIL-15Rβ-hγc complexes. A, The binding activity of IL-15 and IL-15 muteins to c264scTCR/IL-15RαSu-coated wells was determined in an ELISA-based assay. No differences in the binding of rhIL-15 (■), rhIL-15N72D (□), c264scTCR/hIL-15 fusion protein (●) or c264scTCR/hIL-15N72D fusion protein (○) were detected. B & C, Cell-based competitive binding assays were conducted using TF-1 (hIL-15Rα-hγc-positive) (B) or TF-1β(hIL-15Rα-hIL-15Rβ-hγc-positive) (C) cells. Binding of a fixed concentration (500 pM) of c264scTCR/hIL-15 was competed with increasing concentrations of rhIL-15 wild type (■) or rhIL-15N72D (□) proteins. Cell-bound c264scTCR/hIL-15 was then detected by staining with biotinylated anti-human TCR Cβ Ab-PE-conjugated streptavidin followed by flow cytometry analysis. Geo-mean of fluorescence intensity (MFI) was used to calculate the percent binding inhibition at different concentrations of rhIL-15N72D or rhIL-15 protein. The results are representative of at least three experiments.
FIGURE 6
FIGURE 6
Enhanced signal transduction via IL-15R by rhIL-15N72D. TF-1β cells were incubated with medium alone, 10 pM of rhIL-15 or rhIL-15N72D for 15 min at 37°C. Cell extracts were analyzed by Western blotting using anti-phospho-Jak1 (pJak1) antibody (A) or anti-phospho-Stat5 (pStat5) antibody (B). Membranes were then reprobed with antibodies recognizing the native proteins (Jak1 or Stat5). The results are representative of three independent experiments. To correct for possible variation in the amount of protein loaded, values were normalized and are expressed as pJak1/Jak1 or pStat5/Stat5 ratios. pJak1/Jak1 and pStat5/Stat5 levels were determined by densitometry including correction for background. Results are plotted as the mean fold induction (±SEM) in the normalize protein ratios observed in treated compared to untreated cells.
FIGURE 7
FIGURE 7
rhIL-15N72D provides increased anti-apoptotic activity compared to rhIL-15. Apoptosis was evaluated by Annexin V cell-surface expression by flow cytometry. A, Histograms representing Annexin V staining of 32Dβ cells following growth for 48 h in the presence of 361 pM, 22.5 pM, or 0.7 pM of rhIL-15 (gray shade) or rhIL-15N72D (black thick line). B, The percentage of apoptotic cells observed at each concentration of rhIL-15N72D (□) or rhIL-15 wild type (■) protein was plotted and EC50 values determined from the fit curve. The data shown represent the means (±SEM) of three independent experiments.
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