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In situ PEGylation of CAR T cells alleviates cytokine release syndrome and neurotoxicity

Nature Materials (2023)Cite this article

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Abstract

Chimeric antigen receptor T (CAR T) cell immunotherapy is successful at treating many cancers. However, it often induces life-threatening cytokine release syndrome (CRS) and neurotoxicity. Here, we show that in situ conjugation of polyethylene glycol (PEG) to the surface of CAR T cells (‘PEGylation’) creates a polymeric spacer that blocks cell-to-cell interactions between CAR T cells, tumour cells and monocytes. Such blockage hinders intensive tumour lysing and monocyte activation by CAR T cells and, consequently, decreases the secretion of toxic cytokines and alleviates CRS-related symptoms. Over time, the slow expansion of CAR T cells decreases PEG surface density and restores CAR T cell–tumour-cell interactions to induce potent tumour killing. This occurs before the restoration of CAR T cell–monocyte interactions, opening a therapeutic window for tumour killing by CAR T cells before monocyte overactivation. Lethal neurotoxicity is also lower when compared with treatment with the therapeutic antibody tocilizumab, demonstrating that in situ PEGylation of CAR T cells provides a materials-based strategy for safer cellular immunotherapy.

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Fig. 1: PEGylation of CAR T cells alters cell-to-cell interactions and cytokine release.
Fig. 2: Conjugation of DBCO-PEG600K to CAR T cells in vivo.
Fig. 3: In situ PEGylation of CAR T cells alleviates cytokine release syndrome.
Fig. 4: In situ PEGylation of CAR T cells abolishes neurotoxicity.
Fig. 5: In situ PEGylation-induced CRS and neurotoxicity alleviation can also be achieved using the tetrazine (Tz)-TCO reaction.
Fig. 6: Mechanistic illustration of the in situ PEGylation strategy for managing cytokine release syndrome and neurotoxicity.

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All relevant data of this study are available within the paper and its Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

M.J.M. acknowledges support from an NIH Director’s New Innovator Award (no. DP2TR002776), an NSF CAREER Award (no. CBET-2145491), a Burroughs Wellcome Fund Career Award at the Scientific Interface and the American Cancer Society (no. RSG-22-122-01-ET). The authors thank the June lab for the help on CAR T cell preparation. The authors also acknowledge the NIH S10 grant (1S10OD026986) for the support on a cell sorter.

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Author notes

  1. These authors contributed equally: Ningqiang Gong, Xuexiang Han, Lulu Xue.

Authors and Affiliations

  1. Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA

    Ningqiang Gong, Xuexiang Han, Lulu Xue, Rakan El-Mayta, Ann E. Metzloff, Margaret M. Billingsley, Alex G. Hamilton & Michael J. Mitchell

  2. Penn Institute for RNA Innovation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  3. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  4. Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  5. Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  6. Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

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Contributions

N.G. and M.J.M. conceived and designed the experiments. N.G., X.H., L.X. and A.G.H. performed the experiments. N.G., L.X., X.H. and R.E.M. analysed the data. N.G. and M.J.M. wrote the manuscript. M.M.B. and A.E.M. edited the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Michael J. Mitchell.

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N.G. and M.J.M. have filed a patent application related to this study. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Comparison of the CAR T cell ex vivo PEGylation strategy with the in situ PEGylation strategy.

a, Raji tumour-bearing mouse model was constructed, and mice were treated with either ex vivo PEGylated CAR T cells or regular CAR T-azide cells at day 0. On day 1, the mice receiving regular CAR T-azide cells developed high fever (ΔT > 2 °C), after which DBCO-PEG600k (in situ PEGylation) was injected. Mouse body temperature (b), tumour burden (c), blood IL-6 levels (d) and CAR T cell levels (e) were monitored. f, quantification of tumour burden in different groups at day 35. g, Kaplan–Meyer survival plots. Data in (b), (d), (e), and (f) are shown as mean ± s.d. (n = 5). Statistical differences in (b), (d), and (e) were calculated using two-way ANOVA with Tukey’s post hoc test. Statistical differences in (f) were calculated using one-way ANOVA with Tukey’s post hoc test. P values indicated in the figure are from the comparisons on day 7. Statistical differences in (g) were conducted using a Mantel–Cox two-sided log-rank test (n = 5). P values are indicated.

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Gong, N., Han, X., Xue, L. et al. In situ PEGylation of CAR T cells alleviates cytokine release syndrome and neurotoxicity. Nat. Mater. (2023). https://doi.org/10.1038/s41563-023-01646-6

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