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GD2-specific CAR T cells encapsulated in an injectable hydrogel control retinoblastoma and preserve vision


Retinoblastoma (RB) is a pediatric retinal tumor that overexpresses the ganglioside GD2. Although it is treatable in patients with early diagnosis, patients may lose one or two eyes. We generated GD2-specific chimeric antigen receptor T lymphocytes (GD2.CAR-Ts) and locally delivered them to mice with an in situ grafting RB. We showed that, when used in combination with the local release of interleukin-15 and an injectable hydrogel, GD2.CAR-Ts successfully eliminate RB tumor cells without impairing mouse vision.

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Fig. 1: RBs express GD2 on the cell surface, and GD2.CAR-Ts control the growth of RB but fail to prevent tumor recurrence.
Fig. 2: GD2.CAR.15-Ts improve RB treatment but fail to achieve complete tumor eradication.
Fig. 3: Injectable hydrogel further improves treatment with GD2.CAR.15-Ts and prevents tumor recurrence.
Fig. 4: IL-15 and hydrogel prolong the lifespan of GD2.CAR-Ts in tumors.
Fig. 5: GD2.CAR.15-Ts and hydrogel treatment rescue mouse vision in the absence of toxicity to the retina.

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Data availability

BLI and imaging data can be found at figshare ( All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.


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We thank C. Shields and Shields & Shields Oncology (Philadelphia, PA) for providing human RB samples and D. Hill (the University of North Carolina at Chapel Hill) for providing access to a rheometer. We thank R. Hurwitz (Texas Children’s Hospital in Houston) for helpful discussions and comments on the manuscript. We appreciate financial support from the US National Eye Institute (R01EY026564, Z.H.), the Carolina Center of Cancer Nanotechnology Excellence (Z.H.), the NC TraCS Translational Research Grant (550KR151611, Z.H.), the Edward N. & Della L. Thome Memorial Foundation (138289, Z.H.), the BrightFocus Foundation (M2019063, Z.H.), the National Cancer Institute (1R21CA226483-01A1, G.D.) and the University Cancer Research Fund (UCRF, G.D.).

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Authors and Affiliations



K.W., G.D., B.S. and Z.H. designed the project. K.W., Y.C., S.A., M.Z. and E.L. conducted experiments and wrote the manuscript. K.W., Y.C., S.A., M.Z., E.L., B.S. and Z.H. contributed to the protocols and analyzed the data. G.D., B.S. and Z.H. edited the grammar and critically reviewed the manuscript. G.D., B.S. and Z.H. supervised the project. The manuscript was reviewed by all of the authors.

Corresponding authors

Correspondence to Barbara Savoldo or Zongchao Han.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 T cells expressed CAR and effector-tumor coculture exhibited elevated cytokine release.

a, CAR expression in T cells transduced with retroviral vectors encoding GD2.CAR, CD19.CAR, GD2.CAR.IL15, and CD19.CAR.IL15. CAR expression was evaluated by flow cytometry. b, IL-2 and IFN-γ released in the culture supernatants of CAR-Ts co-cultured with tumor cells. Supernatant was collected at 24 hours and cytokines were measured by specific ELISA (E:T=1:5, n = 5 donor/group, E:T=1:2, n = 3 donor/group). Data presented show mean and SEM.

Source data

Extended Data Fig. 2 The morphologic and rheologic properties of Chitosan-PEG hydrogel.

a, Lyophilized injectable hydrogel was imaged by scanning electron microscopy. The image showed 63 um average pore diameter of the hydrogel (n=10 technical replicates). b, GD2.CAR-Ts labelled with eGFP-FFLuc were encapsulated into the hydrogel and visualized by florescence macroscopy (B). Assessment of the viscoelastic properties of the injectable hydrogel tested at 23 °C and 37 °C (n=10 technical replicates) c, Rheological test result of the chitosan-PEG injectable hydrogel and water/saline16 (n=10 technical replicates). Data presented show mean and SD.

Extended Data Fig. 3 Molecular characterization of chitosan-PEG hydrogel.

a, 1H NMR spectra of the chitosan and the chitosan-PEG hydrogel precursor and (b) molecular weight of Chitosan-PEG precursors measured by dynamics light scattering (n=12 technical replicates). Data presented show mean and SD.

Extended Data Fig. 4 CAR-Ts release from hydrogels and chitosan-PEG hydrogel encapsulated GD2.CAR-Ts effectively eliminated tumor.

a, Viability of CAR-Ts released from alginate-gelatin and chitosan-PEG injectable hydrogel (n =3 donor/group). b, CAR-T cell release profile from alginate-gelatin and chitosan-PEG injectable hydrogel (n =3 donors/group). RB tumor cell lines were co-cultured with GD2.CAR-Ts or CD19.CAR-Ts at the effector to tumor ratio of 1:1 (c) and 1:5 (d) for 7 days upon encapsulation with the hydrogels. All cells were collected and analyzed by flow cytometry to enumerate T cells and residual tumor cells in the culture (n =3 donors/group). Data presented show mean and SEM.

Source data

Extended Data Fig. 5 The hydrogel facilitates the antitumor activity of GD2.CAR-Ts.

a, Migration assay showing T cell migration and viability in the collagen gel (Fluorescence Green: Living cells, Red: Dead cells) on days 1 and 4.24 b, Western blotting analysis of the protein lysate of the dissected eyes at day 35 post-treatment to detect IL-15. The statistical significance was determined using one-way ANOVA with Tukey’s post hoc test (GD2.CAR.15 versus GD2.CAR.15 hydrogel) P=0.012. n=3 individual sample/group. Data presented as mean and SEM.

Source data

Extended Data Fig. 6 The Gating strategy and photography of mice.

a, Gating strategy to assess effector-tumor coculture (CD4+/CD8+ cells) and the presence of T cells (CD45+ cells) and RB tumor cells (GD2+ cells) in the posterior segment of the eye by flow cytometry. b, Photographs of representative nu/nu mice (n=15/group) 35 days after engraftment with the Y79-eGFP-FFLuc tumor cell line and receiving the indicated treatments.

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Wang, K., Chen, Y., Ahn, S. et al. GD2-specific CAR T cells encapsulated in an injectable hydrogel control retinoblastoma and preserve vision. Nat Cancer 1, 990–997 (2020).

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