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Suppression of cytokine release syndrome during CAR-T-cell therapy via a subcutaneously injected interleukin-6-adsorbing hydrogel

Nature Biomedical Engineering volume 7, pages 1129–1141 (2023)Cite this article

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Abstract

The infusion of chimaeric antigen receptor (CAR) T cells can trigger the release of life-threatening supraphysiological levels of pro-inflammatory cytokines. However, uncertainty regarding the timing and severity of such cytokine release syndrome (CRS) demands careful monitoring of the conditions required for the administration of neutralizing antibodies. Here we show that a temperature-sensitive hydrogel conjugated with antibodies for the pro-inflammatory cytokine interleukin-6 (IL-6) and subcutaneously injected before the infusion of CAR-T cells substantially reduces the levels of IL-6 during CRS while maintaining the therapy’s antitumour efficacy. In immunodeficient mice and in mice with transplanted human haematopoietic stem cells, the subcutaneous IL-6-adsorbing hydrogel largely suppressed CAR-T-cell-induced CRS, substantially improving the animals’ survival and alleviating their levels of fever, hypotension and weight loss relative to the administration of free IL-6 antibodies. The implanted hydrogel, which can be easily removed with a syringe following a cooling-induced gel–sol transition, may allow for a shift in the management of CRS, from monitoring to prevention.

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Fig. 1: IL6S prevents CAR-T-cell-induced cytokine-release storm and related adverse effects.
Fig. 2: Synthesis and characterization of IL6S.
Fig. 3: The adsorption performance and mechanism of IL6S.
Fig. 4: IL6S absorbs IL-6 in a real-time manner.
Fig. 5: IL6S suppresses CAR-T-cell-induced CRS in a SCID-beige mouse model.
Fig. 6: IL6S suppresses CAR-T-cell-induced CRS in a humanized NCG-SGM3 mouse model.
Fig. 7: Ice coating-assisted removal of IL6S and antitumour efficacy of CAR-T cells in vivo.

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Data availability

The data supporting the results in this study are available within the paper and its Supplementary Information. All data generated in this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (grant no. 2021YFA1201000), National Natural Science Foundation of China (NSFC) key project grant nos. 32030060 and 81773185, and NSFC international collaboration key project grant no. 51861135103. We also appreciate the support of the Chinese Academy of Sciences (CAS-NSTDA) International Partnership Program (121D11KYSB20210003).

Author information

Author notes

  1. These authors contributed equally: Xianlei Li, Ningqiang Gong.

Authors and Affiliations

  1. Laboratory of Controllable Nanopharmaceuticals, Chinese Academy of Sciences (CAS) Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, P. R. China

    Xianlei Li, Ningqiang Gong, Yuxuan Zhang, Yufei Wang, Guangchao Qing, Yongchao Wang, Fangzhou Li, Jinjin Wang, Qiankun Ni, Yaling Gan, Yan Wu & Xing-Jie Liang

  2. University of Chinese Academy of Sciences, Beijing, P. R. China

    Xianlei Li, Ningqiang Gong, Falin Tian, Yuxuan Zhang, Yufei Wang, Jinjin Wang, Qiankun Ni, Xinghua Shi, Yan Wu & Xing-Jie Liang

  3. CAS Key Laboratory for Nanosystem and Hierarchy Fabrication, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, P. R. China

    Falin Tian & Xinghua Shi

  4. Institute of Biology and Medicine, Wuhan University of Science and Technology, Wuhan, Hubei, P. R. China

    Shangkun Zhang, Chaojiang Gu & Tongcun Zhang

  5. Center for Transformative Science, ShanghaiTech University, Shanghai, P. R. China

    Yihui Xu & Huaidong Jiang

  6. School of Life Science and Technology, ShanghaiTech University, Shanghai, P. R. China

    Linlin Zhang & Xingxu Huang

  7. School of Biomedical Engineering, Guangzhou Medical University, Guangzhou, Guangdong, P. R. China

    Xing-Jie Liang

Authors
  1. Xianlei Li
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Contributions

X.L., N.G., Y. Wu and X.-J.L. designed the project and experiments, analysed the experimental data and wrote the manuscript. X.L., N.G., Y.Z., Yufei Wang, G.Q., Yongchao Wang, F.L., J.W. and Q.N. carried out all experiments. X.S. and F.T. guided and performed computational simulation. T.Z., X.H., C.G., S.Z. and L.Z. guided the construction and culture of CAR-T cells. Y.G., H.J. and Y.X. guided materials morphology characterization. X.L., N.G., Y. Wu and X.-J.L. supervised the entire project.

Corresponding authors

Correspondence to Ningqiang Gong, Yan Wu or Xing-Jie Liang.

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Nature Biomedical Engineering thanks Zhen Gu, Jamal Lewis 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 The evaluation of the biocompatibility of IL6S and the effect of IL6S on CAR-T-cell-induced tumour cell killing.

The viability of CAR-T cells (a), human umbilical vein endothelial cells (HUVEC) (b), and mouse embryonic fibroblasts cells (NIH3T3) (c) after treatment with IL6S (n = 5). Cells were incubated with different concentrations of IL6S for 24 h before viability was determined. d, Hemolytic analysis of red blood cells exposed to different concentrations of IL6S. e, The impact of IL6S on CAR-T-cell-induced tumour cell killing was assessed by flow cytometry and confocal fluorescence imaging (inset). The red dotted boxes indicate live Raji cells in different groups. f, The effect of IL6S on CAR-T-cell-induced tumour cell killing was determined by measuring luciferase expression from live Raji-Luc cells. g, Quantification of the luminescent signals of Raji-Luc cells after different treatments (n = 5). Data are presented as mean ± s.d. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparison was used for the calculation of the statistical differences in (a), (b) and (c). Two-sided unpaired Student’s t-test was used for the calculation of the statistical differences in (g). There is no significant (NS) difference between groups when the P value is higher than 0.05. NS, P > 0.05, *P < 0.05, **P < 0.01.

Source data

Extended Data Fig. 2 IL6S decreases IL-6 in the serum and major organs in a CRS-mimicking mouse model.

The CRS-mimicking mouse model was constructed through i.v. injection of 10 ng IL-6 to healthy SCID-beige mouse. The experimental groups were set as follows: Control, healthy mice without any treatment; IL-6, intravenous injection of IL-6 for mimicking CRS; IL-6&IL6S, a primary subcutaneous injection of IL6S followed by an intravenous injection of IL-6. The serum and major organs were collected 24 hours after the IL-6 and IL6S administration. The IL-6 levels in serum (a), heart (b), liver (c), spleen (d), lung (e), kidney (f) and brain (g) were measured using an IL-6 ELISA kit. Data are presented as mean ± s.d. (n = 3). Two-sided unpaired Student’s t-test was used for the calculation of the statistical differences in (a), (b), (c), (f) and (g). Two-sided Welch’s t-test was used for the calculation of the statistical differences in (d). There is no significant (ns) difference between groups when the P value is higher than 0.05. nsP > 0.05, *P < 0.05, **P < 0.01.

Source data

Extended Data Fig. 3 IL6S decreases the concentration of multiple cytokines in the serum of CAR-T-cell-induced CRS mouse model.

a, Schematic showing the process of CRS model construction and the treatment of mice with free antibodies or IL6S. 2 days post-injection of CAR-T cells, the serum of all mice was collected for multi-cytokine measurements. The concentrations of murine IL-6 (b), CCL2 (c), SAA3 (d), G-CSF (e) and CXCL9 (f), human GM-CSF (g) and IL-3 (h) in mouse serum collected from the six treatment groups. Data are presented as mean ± s.d. (n = 5). Two-sided Welch’s t-test was used for the calculation of the statistical differences in (b), (d), (e) and (g). There is no significant (ns) difference between groups when the P value is higher than 0.05. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 4 IL6S decreases IL-6 levels in major organs in a CAR-T-cell-induced CRS model.

A Raji tumour-bearing mice model was constructed and the mice were s.c. injected with IL6S before CAR-T cell injection. After 48 h, mice were euthanized and the IL-6 levels in heart (a), liver (b), spleen (c), lung (d), and kidney (e) were examined. Control group: healthy mice without any treatment. CRS group: tumour-bearing mice treated with CAR-T cells. CRS&IS group: mice were treated with s.c. injection of 100 μL IL6S (hydrogel concentration = 100 mg/mL, IL-6 antibody concentration = 1 ng/μL) and followed by CAR-T cell injection. f, Representative immunofluorescence images of mouse major organs from the CRS&IL6S group. Green, F4/80; red, CD80; blue: nucleus. (Scale bar: 100 μm). Data in a-e are presented as mean ± s.d. (n = 3). Two-sided unpaired Student’s t-test was used for the calculation of statistical differences in (a), (c), (e), and group “Control” versus “CRS&IL6S” in (d). Two-sided Welch’s t-test was used for the calculation of statistical differences in group “CRS” versus “CRS&IL6S” in (d). There is no significant (ns) difference between groups when the P value is higher than 0.05. nsP > 0.05, *P < 0.05, ***P < 0.001, ****P < 0.0001.

Source data

Extended Data Fig. 5 IL6S ablates the accumulation of multiple cytokines in the mouse brain.

a, Schematic showing the process of CRS model construction and the treatment with free antibodies or IL6S. 2 days post-injection of CAR-T cells, brain tissue of mice was collected for multi-cytokine measurements. The concentrations of murine IL-6 (b), CCL2 (c), SAA3 (d), G-CSF (e) and CXCL9 (f), human GM-CSF (g) and IL-3 (h) in brain tissue. Data are presented as mean ± s.d. (n = 5). Two-sided Welch’s t-test was used for the calculation of the statistical differences in (b), (d), (e) and (g). There is no significant (ns) difference between groups when the P value is higher than 0.05. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Supplementary information

Supplementary Information

Supplementary figures, tables and references.

Reporting Summary

Peer Review File

Supplementary Video 1

Fluorescent 3D reconstruction of the sponge frame and antibody distribution.

Supplementary Video 2

IL-6 adsorption process at 0.2x.

Supplementary Video 3

IL-6 adsorption process at 1x.

Supplementary Video 4

IL-6 adsorption process at 3x.

Supplementary Video 5

Dynamic interaction of IL-6 and the conjugated antibody.

Supplementary Table 1

Statistical analyses.

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Li, X., Gong, N., Tian, F. et al. Suppression of cytokine release syndrome during CAR-T-cell therapy via a subcutaneously injected interleukin-6-adsorbing hydrogel. Nat. Biomed. Eng 7, 1129–1141 (2023). https://doi.org/10.1038/s41551-023-01084-4

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