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Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells

Nature Biomedical Engineering volume 5, pages 1288–1305 (2021)Cite this article

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Abstract

The preferential activation of regulatory T (Treg) cells by interleukin-2 (IL-2), which selectively binds to the trimeric IL-2 receptor (IL-2R) on Treg cells, makes this cytokine a promising therapeutic for the treatment of autoimmune diseases. However, IL-2 has a narrow therapeutic window and a short half-life. Here, we show that the pharmacokinetics and half-life of IL-2 can be substantially improved by orthogonally conjugating the cytokine to poly(ethylene glycol) (PEG) moieties via a copper-free click reaction through the incorporation of azide-bearing amino acids at defined sites. Subcutaneous injection of a PEGylated IL-2 that optimally induced sustained Treg-cell activation and expansion over a wide range of doses through highly selective binding to trimeric IL-2R led to enhanced therapeutic efficacy in mouse models of lupus, collagen-induced arthritis and graft-versus-host disease without compromising the immune defences of the host against viral infection. Site-specific PEGylation could be used more generally to engineer cytokines with improved therapeutic performance for the treatment of autoimmune diseases.

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Fig. 1: Site-specific PEGylation of human IL-2 mediated by amber codon suppression and screening of resultant PEGylated variants with differential binding to the α or β subunit of IL-2R for identification of receptor-biased IL-2 ligands showing the highest level of selectivity to trimeric IL-2Rs.
Fig. 2: Profiling of the receptor-biased IL-2 PEGylates in terms of selective activation of Treg cells versus CD8+ T cells in vitro.
Fig. 3: Evaluation of the effects of PEGylation on the pharmacokinetic properties of IL-2.
Fig. 4: Prevention of disease aggravation in GvHD mice by PEGylated IL-2 variants through selective Treg-cell proliferation and activation.
Fig. 5: Dual-31/51-20K ameliorated SLE in MRL/lpr mice and ex vitro preferential activation of human Treg cells from patients with SLE.
Fig. 6: Dual-31/51-20K-mediated amelioration of CIA.
Fig. 7: Assessment of the impact of IL-2 PEGylation on the immune responses of mice to the influenza virus.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are too large to be publicly shared, yet they are available for research purposes from the corresponding authors on reasonable request. The quaternary structure of IL-2 associated with the trimeric receptor is available from publicly available datasets (http://www1.rcsb.org).

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Acknowledgements

We thank Z. Tian (University of Science and Technology of China, Hefei) for gifting the YT cell line; Y. Hou for her support with flow cytometry technology and D. Liu (Peking University) for her participation in mass spectrometry experiments; X. Wu from the X.Z. laboratory and H. Li from the Tsokos laboratory for their discussions; and all of the donors who participated in the study. This study was supported by grants from the National Key Research and Development Program of China (no. 2019ZX09739), National Natural Science Foundation of China (nos 81788101, 81803419, 81630044, 81802121, 21805311), Chinese Academy of Medical Science Innovation Fund for Medical Sciences (nos CIFMS2016-12M-1-003, 2017-12M-1-008, 2017-I2M-3-011, 2016-12M-1-008) and Capital’s Funds for Health Improvement and Research (no. 2020-2-4019).

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Author notes

  1. These authors contributed equally: Bo Zhang, Jiaqi Sun, Yan Wang, Dezhong Ji, Yeshuang Yuan.

Authors and Affiliations

  1. State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Bo Zhang, Shengjie Li, Yeting Sun, Pengchong Li, Lidan Zhao, Jin Hu, Wei Song, Xiaogang Li, Yunyun Fei & Hua Chen

  2. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China

    Bo Zhang, Jiaqi Sun, Yan Wang, Dezhong Ji, Fei Yu, Wenxiao Ma, Boyang Cheng, Ling Wu, Lihe Zhang & Demin Zhou

  3. Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Yeshuang Yuan & Min Wang

  4. Department of Rheumatology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Clinical Immunology Center, Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Yeshuang Yuan, Min Wang & Xuan Zhang

  5. The Ministry of Education Key Laboratory, Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Yeting Sun, Pengchong Li, Lidan Zhao, Yunyun Fei & Hua Chen

  6. Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science and Engineering, Peking University, Beijing, China

    Yingqin Hou

  7. Division of Rheumatology and Clinical Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Hao Li & George C. Tsokos

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  1. Bo Zhang
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Contributions

B.Z., Y.W., J.S., D.Z. and X.Z. designed the study. B.Z., Y.W., J.S., Y.H., J.H., Y.Y. and Y.S. performed the experiments in vitro and in vivo and analysed the data. D.J. and F.Y. performed experiments of chemical synthesis. S.L. contributed to the crystal structure analysis and site selection. W.M. and B.C. performed experiments with influenza virus. P.L., L.W., W.S., M.W. and X.L. contributed to the collection and assembly of data and polished the language. L. Zhao, H.C., Y.F., H.L., L. Zhang and G.C.T. provided comments. B.Z., D.Z., G.C.T. and Z.X. wrote and reviewed the manuscript. All of the authors reviewed the manuscript.

Corresponding authors

Correspondence to George C. Tsokos, Demin Zhou or Xuan Zhang.

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Peer review information Nature Biomedical Engineering thanks Onur Boyman, David Klatzmann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Characterization of the binding of representative PEGylated variants to dimeric or trimeric IL-2Rs, assessed by surface plasmon resonance (SPR), for identification of IL-2 PEGylates showing the highest level of selectivity towards trimeric IL-2Rs.

Fc-tagged extracellular domains of the IL-2R subunits were immobilized on a CM5 chip at an equimolar ratio. Three representative concentrations of each sample in a series of 3-fold dilutions for trimeric (3.3, 10 and 30 nM) or dimeric (33.3, 100 and 300 nM) IL-2R complexes are presented. All kinetic parameters are provided in S Table 2. Representative results from one of two independent experiments are shown. See also Fig. 1.

Extended Data Fig. 2 Validation of Treg-selective activation, as reflected by increased expression of CD25 and FoxP3 by PEGylated IL-2s in a xeno-GvHD mouse model.

The MFI bars graphs (top) and flow cytometry histograms (bottom) of induced CD25 and Foxp3 on Tregs and CD8 + T cells in response to IL-2 and its PEGylated variants at a dose of 2 µg, with PBS as the background. The data are presented as the mean ± SEM, n = 3 biologically independent mice per group. Statistical analysis was performed using one-way ANOVA (Dunnett’s multiple-comparison test compared with IL-2 treated group). Representative results from one of three independent experiments are shown. See also Fig. 4a, b.

Extended Data Fig. 3 Dual-31/51-20K-mediated mitigation of kidney lesions in MRL/lpr mice.

The ability of dual-31/51-20 K to reduce kidney lesions, as reflected by H&E staining of the 0.5 µg treatment groups, with the mononuclear cells infiltrating into the cortical tubulointerstitial/perivascular areas and the scores of glomerular and perivascular lesions shown by arrows and bar graphs, respectively. The data are presented as the mean ± SEM, n = 4 biologically independent mice per group. Statistical analysis was performed using one-way ANOVA (Dunnett’s multiple-comparison test compared with PBS treated group). Representative results from one of two experiments are shown. See also Fig. 5b, c.

Extended Data Fig. 4 The effects of IL-2 versus dual-31/51-20 K on activation of pulmonary NK cells.

Bar graphs show the expression of activated markers, including NKG2D, NKp46, and CD69, on pulmonary NK cells of mice with indicated treatment. Data are presented as the mean ± SEM, n = 4 biologically independent mice per group. Statistical analysis was performed using one-way ANOVA (Dunnett’s multiple-comparison test compared with + virus/PBS-treated group). The experiment was repeated three times with similar results, and one of three representative results is shown. See also Fig. 7c.

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Zhang, B., Sun, J., Wang, Y. et al. Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells. Nat Biomed Eng 5, 1288–1305 (2021). https://doi.org/10.1038/s41551-021-00797-8

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