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.
RB is a pediatric cancer that develops within the retina and occurs in either a heritable form, in association with germline mutations in RB1 (encoding the RB transcriptional corepressor 1), or, more frequently, in a nonheritable form1. When RB is confined to the eye, cryotherapy, thermotherapy, chemotherapy and radiotherapy are used, but enucleation, with consequent loss of vision, is frequently the best option for advanced stages2,3. Development of treatment strategies that can eradicate the tumor but preserve vision would significantly enhance the quality of life of patients with RB. Immunotherapy in the form of adoptive transfer of CAR-Ts is perceived as the most promising therapy for pediatric patients with acute lymphoblastic leukemia4. Here we developed a local immunotherapy approach based on GD2-specific CAR-Ts releasing interleukin (IL)-15 and encapsulated in an in situ-formed injectable chitosan hydrogel that allows tumor eradication while preserving vision.
RB, like other neuroepithelial cancers, expresses the ganglioside GD2 (ref. 5). GD2 has been targeted in pediatric patients with neuroblastoma (NB) with either monoclonal antibodies or more recently with CAR-Ts6,7. In particular, GD2.CAR-Ts were well tolerated and produced clinical benefits in some patients with NB6. We evaluated GD2 expression in human RB specimens. GD2 was expressed in human RB, but not in normal human retina (Fig. 1a). GD2 was also highly expressed in the two RB cell lines Y79 and WERI-Rb-1 (Fig. 1b). We then investigated whether GD2.CAR-Ts could target RB cancer cells by co-culturing CAR-Ts with RB tumor cell lines. Both CD19-specific CAR (CD19.CAR, the control) and GD2.CAR were expressed in transduced T cells (Extended Data Fig. 1a), but only GD2.CAR-Ts specifically eliminated both Y79 and WERI-Rb-1 RB cells (Fig. 1c). The antitumor activity of GD2.CAR-Ts was corroborated by the detection of T helper type 1 (TH1) cytokines (interferon (IFN)-γ and IL-2) in culture supernatants, as well as by their proliferation in response to RB tumor cells (Extended Data Fig. 1b).
To evaluate the antitumor effects of GD2.CAR-Ts in vivo, we used an in situ xenograft nude mouse model as previously described8. In this model, RB tumor cells are implanted in the subretinal space, creating a tumor that mimics the human disease, which better recapitulates the origin of human RB than intravitreal inoculation. We stably expressed (via a retroviral vector) an enhanced GFP (eGFP)–firefly luciferase (FFLuc) fusion in the Y79 RB cell line and engrafted 104 cells via subretinal injection. Seven days later, GD2.CAR-Ts or CD19.CAR-Ts (106 cells) were inoculated intratumorally, via subretinal injection. GD2.CAR-Ts promoted delayed tumor growth, measured by either bioluminescence (BLI) or direct tumor measurement (Fig. 1d,e,g), but did not cause tumor eradication, as all mice developed tumors and required euthanasia by day 70 (Fig. 1f,g). Overall, these data suggest that local injection of GD2.CAR-Ts can transiently control RB growth.
In attempting to eradicate the tumor, we hypothesized that providing GD2.CAR-Ts with self-growth factor support would enhance their therapeutic effect by prolonging their persistence within the tumor. We introduced IL-15, an immune-enhancing cytokine known to promote T cell proliferation, activation and survival9, into the GD2.CAR construct10 (Extended Data Fig. 1a). While the addition of IL-15 did not cause RB regression in mice treated with CD19.CAR-Ts, GD2.CAR-Ts releasing IL-15 (GD2.CAR.15) showed significantly improved antitumor effects, with 60% of treated mice remaining tumor free up to day 70 (Fig. 2a–e; P = 0.0010). These data indicate that IL-15 enhances the antitumor effects of GD2.CAR-Ts by sustaining their local survival.
Intratumor delivery of T cells occurs as a single ‘all-at-once’ delivery and does not allow for the extended time of intralesional persistence of CAR-Ts that is required to sustain antitumor effects. A delivery vehicle promoting CAR-T persistence would be highly preferable. In situ-formed injectable hydrogels allow for localized and sustained cell delivery11,12,13. We selected the chitosan–polyethylene glycol (PEG) thermosensitive hydrogel, formed at physiological temperature, as the optimal injectable hydrogel to deliver CAR-Ts within the eye (Extended Data Figs. 2 and 3). The majority of T cells encapsulated within the hydrogel remained viable, were released from the gel in vitro within 1 week and retained their cytotoxic activity (Extended Data Fig. 4). We next tested whether the encapsulated GD2.CAR.15-Ts would eradicate RB. The chitosan–PEG hydrogel significantly improved the antitumor effects of GD2.CAR-Ts (Fig. 3a; P = 0.0024, n = 15), but the combination of the GD2.CAR.15-Ts and the chitosan–PEG hydrogel completely controlled tumor growth and prevented tumor recurrence (P < 0.0001, n = 15; Fig. 3b–d). Histological studies confirmed that the combination of injectable hydrogel and GD2.CAR-Ts or GD2.CAR.15-Ts not only allowed tumor clearance, but also improved structural recovery of the retina (Fig. 3e). Analysis of the dissected posterior segments at day 60 post-treatment showed the presence of GD2.CAR-Ts in mice engrafted with the combination of hydrogel and GD2.CAR-Ts secreting IL-15 (Fig. 4a,b). The retinal structure was essentially preserved, even though signs of an inflammatory response and a cavity corresponding to the eliminated tumor were observed (Fig. 4c). In addition, the hydrogel facilitated migration of T cells and further improved their viability. Western blotting of lysates obtained from the eyes of treated mice further revealed that the hydrogel increased IL-15 detection within the eye. This effect could be due to an enhanced persistence of T cells and/or an increased retention of IL-15 within the eye (Extended Data Fig. 5a,b). Overall, these data support the conclusion that local co-injection of the chitosan–PEG thermosensitive hydrogel and GD2.CAR.15-Ts eradicates RB.
To further evaluate whether the proposed local immunotherapy preserves retinal function, we performed fundoscopy, optical coherence tomography (OCT) and electroretinography (ERG) analyses. The fundus imaging revealed morphological changes under the retina after tumor engrafting and treatment8. After Y79-eGFP–FFLuc tumor cell engraftment, the GFP signal was clearly detectable in control mice, but absent in mice treated with the combination of hydrogel and GD2.CAR-Ts secreting IL-15, without evidence of macroscopic damage to the retina (Fig. 5a). ERGs performed to test the retinal response to light showed a loss of retinal response in tumor-bearing mice, as evidenced by a decrease in both scotopic a and b waves and the photopic b wave (Fig. 5b). In contrast, in mice treated with the combination of hydrogel and GD2.CAR.15-Ts, retinal function was significantly improved (P = 0.009, n = 7; Fig. 5b).
Here we propose a local immunotherapy approach that combines effector T cells targeting GD2, the T cell growth factor IL-15 and a chitosan–PEG injectable hydrogel to eradicate RB in a preclinical model. GD2 is a marker on neuroepithelial tumors, and GD2-specific antibodies and CARs have been developed6,7,14. However, to date, no attempt to target GD2 in RB has been reported. GD2.CAR-Ts showed potent cytotoxic activity against RB tumor cell lines in vitro. However, GD2.CAR-Ts eradicated RB in vivo only upon the incorporation of IL-15 and encapsulation within chitosan–PEG injectable hydrogel, which improve T cell lifespan and biodistribution, respectively. While we previously reported the critical role of IL-15 expressed by CAR-Ts in prolonging their survival and antitumor activity in metastatic tumor models10,15, we report here the critical role of a biomaterial scaffold in supporting the antitumor effect of CAR-Ts when injected intratumorally.
Chitosan-based injectable hydrogels are biocompatible and biodegradable13,16,17. Previous studies using the chitosan–PEG hydrogel demonstrated its positive effects on cell delivery due to its low viscosity13,18. The in situ gelation we propose facilitates CAR-T movements and confines IL-15 within the hydrogel, thus facilitating use of the cytokine by the CAR-Ts and limiting its diffusion. Remarkably, the immunotherapy approach we propose preserves retinal function. While activated T cells may cause toxicity in the retina, the injectable hydrogel reduced inflammation and retinal detachment by localized delivery of T cells. The proposed approach has the potential for clinical translation as GD2.CAR-Ts and GD2.CAR-natural killer T cells releasing IL-15 are currently being tested in clinical trials in patients with NB (NCT03721068 and NCT03294954), although the safety of intratumoral injection will be further evaluated in a future study of RB immune therapy. Furthermore, the injectable chitosan–PEG hydrogel can be generated by following good laboratory practices or good manufacturing practices11,16.
Synthesis and characterization of injectable hydrogels
The chitosan–PEG hydrogel was synthesized as previously described17,19. PEG (0.2 g) and N-hydroxysuccinimide (NHS, 0.2 g) were dissolved in 5 ml of DMSO and reacted for 72 h at room temperature. The solvent was then removed using evaporation. Thereafter, chitosan was dissolved in a 2% (wt/vol) acetic acid aqueous solution to a final concentration of 2% (wt/vol). The reaction product (NHS–PEG–NHS) was mixed with the 2% (wt/vol) chitosan solution. The reaction was carried out for 24 h, and the product was purified via centrifugal ultrafiltration (10-kDa molecular weight cutoff) by buffer exchange with deionized water. Finally, the product was mixed with a β-glycerophosphate solution to finalize the hydrogel precursor for injection. Alginate hydrogel synthesis was performed by following the procedure in our previous study20. Briefly, 5 ml of a 2% alginate aqueous solution was mixed with 5 ml of a 2% gelatin aqueous solution in a 20-ml vial. Next, 40 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 30 mg NHS were added and reacted at ambient temperature for 24 h. The hydrogel precursor was mixed with the same volume of a 0.05 M CaCl2 solution spontaneously to finish cross-linking. The structure of the hydrogel was characterized by 1H NMR spectroscopy (600-MHz Inova spectrometer, D2O solvent), and the morphology was observed by scanning electron microscopy (Zeiss Supra 25). The rheological test was performed by using a discovery HR-1 rheometer (TA Instruments) with a 20-mm-diameter parallel plate at ambient and physiological temperatures. The linear viscoelastic region was determined by performing a strain sweep from 0.1% to 10% strain at an oscillation frequency of 1 Hz. The storage and loss modulus were calculated using a linear range (0–5% strain) average.
Cell culture, production of CAR-Ts and functional assays
All cell lines (Y79 and WERI-Rb-1) used in this study were purchased from the American Type Culture Collection. Y79 and WERI-Rb-1 cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% GlutaMAX. Generation of GD2.CAR-Ts and GD2.CAR.15-Ts was performed by following the same procedure as previously described10. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats (Gulf Coast Regional Blood Center) using Lymphoprep (Accurate Chemical & Scientific). PBMCs were then activated using 1 mg ml−1 immobilized antibodies to CD3 (Miltenyi Biotec) and CD28 (BD Biosciences). After 3 d, cells were transduced with retroviral supernatants and expanded in complete medium supplemented with IL-7 (10 ng ml−1, PeproTech) and IL-15 (5 ng ml−1, PeproTech) and were further expanded by feeding every 3 to 4 d with fresh complete medium and cytokines21,22. The expression of the CAR was analyzed by following a previously described procedure10. Tumor cell immunophenotyping was performed by following a previously described procedure10. Briefly, Y79 and WERI-Rb-1 cells were harvested and washed with FACS buffer. Next, the tumor cells were incubated with anti-GD2 antibody (BD Biosciences, clone 14.G2a) or an isotype control for 30 min at 4 °C in the dark. Finally, the cells were washed with FACS buffer and suspended in FACS buffer for flow cytometry analysis on a BD LSRFortessa cell analyzer with FACSDiva software. Flow cytometry data were processed with FCS Express 6 software. The gating strategy is shown in Extended Data Fig. 6a. The isotype control was used to gate negative samples. The staining methods were described previously; anti-CD3–APC, anti-CD4–FITC, anti-CD8–PE, anti-CD45–BV510 and anti-GD2–BV711 antibodies were used. Counting beads (CountBright, Invitrogen) were added by following the manufacturer’s instructions.
T cell release from the hydrogel and functional activity
The alginate and chitosan hydrogels were encapsulated in a 0.2-ml volume with 1 × 106, 4 × 106 or 1 × 107 T cells. The hydrogel was formed in a six-well Transwell plate with culture medium. Cell counts and cell viability were assessed daily using trypan blue. To measure the antitumor activity of CAR-Ts released from the hydrogel, 2.5 × 105 Y79 or WERI-Rb-1 cells were seeded in a 24-well plate before adding hydrogel. T cells were encapsulated with alginate–gelatin and chitosan–PEG hydrogels in 0.2-ml volumes with 0.5 × 105 or 2.5 × 105 T cells before being transferred to each well. Blank hydrogel and T cells without hydrogel were used as controls. After co-culturing for 72 h, tumor cells were imaged by fluorescence microscopy and enumerated by flow cytometry. Cytokines (IL-2 and IFN-γ) that are released by CAR-Ts upon targeting tumor cells were measured in the coculture supernatant collected after 24 h, using specific ELISA assays (R&D Systems).
In vivo studies
Animal experiments were conducted in accordance with the policies of the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill. Male and female 6- to 8-week-old NU/NU nude mice were purchased from Charles River Laboratory. Ketamine (100 mg per kg) and xylazine (10 mg per kg) injected intraperitoneally or inhalation of 2.5% isoflurane was used for anesthesia. To establish the RB xenograft model, 1 × 104 tumor cells suspended in a final volume of 1 µl were injected under the retina8. Seven days later, 1 × 106 CAR-Ts in PBS or encapsulated in 1 µl of hydrogel were also injected under the retina. Blank hydrogels were also used as controls. Tumor growth was monitored by BLI using a Caliper IVIS imaging system. Mice were infused with 150 mg per kg body weight d-luciferin 10 min before imaging. The exposure time was 1 or 5 min, and BLI was determined using Living Image software. Mice were killed when the tumor invaded the cornea or when the tumor volume exceeded 500 mm3. Photographs of mice are shown in Extended Data Fig. 6b.
Fundus and OCT imaging
Mice were killed and their pupils were dilated with 1% tropicamide. Fundus images were captured using a Micron IV retinal imaging system (Phoenix Research Labs). Corneas were moistened with Genteal lubricant eye gel (Novartis) and positioned with the Micron eyepiece in direct contact with the eye through the gel. OCT images were captured by using the full-scan setting at an average of ten frames per scan.
ERG was recorded and analyzed according to previously published protocols23. Briefly, mice were adapted to the dark overnight and their eyes were dilated with 1% tropicamide under dim red light. Genteal was applied to each eye, and recordings were made using gold electrodes that were placed on the cornea. The reference electrode was inserted subcutaneously behind the head, and the ground electrode was inserted in the tail. ERG data were collected using an Espion E2 system (Diagnosys), and mouse body temperature was maintained at 37 °C during the experiment.
T cell migration and IL-15 retention assay
The migration of GD2.CAR.15 CAR-Ts was measured by placing 106 T cells in a chitosan–PEG hydrogel or PBS into a three-dimensional collagen gel (2 mg ml−1, Sigma) in complete medium24. T cells migrated through the collagen–gel boundary, were stained with a LIVE/DEAD viability kit (Invitrogen) and were imaged with fluorescence microscopy. The migration distance was measured using ImageJ software.
Histology sectioning, immunohistochemistry and western blotting
Human RB tumors were frozen in optimal cutting temperature compound and serially sectioned (10 µm). Slides were fixed with 4% paraformaldehyde for 10 min (−20 °C) and immunohistochemical analyses were performed. RB tumors were detected with an anti-GD2 antibody (1:400, MilliporeSigma). AF488 donkey anti-mouse antibody (Life Technologies, 1:500) was used as secondary antibody. Omission of the primary antibody was used as a negative control for nonspecific binding. Cell nuclei were stained for 10 min with DAPI (1:5,000) for confocal microscopy. Mice were randomly selected from each cohort and killed for histology and western blotting analysis 35 d post-treatment. H&E staining of eye tissues was carried out by the UNC Center for Gastrointestinal Biology and Disease Histology Core Facility. Briefly, fixed tissues were embedded in paraffin and sectioned into 10-μm sections for eye tissues. Sections were stained with H&E according to conventional staining protocols and visualized using conventional bright-field microscopy. Mouse eyes were dissected. Lysates obtained from the dissected eye were used for western blotting. Lysates were loaded in sample buffer and separated using 4–20% SDS–PAGE gels (Bio-Rad). Gels were transferred onto PVDF blotting membranes, stained with an anti-IL-15 antibody (Prosci, 1:500) and imaged according to the manufacturer’s (Bio-Rad) instructions.
Statistics and reproducibility
All quantitative data are expressed as mean and s.d. The size of animal samples was calculated based on GPower 7.1 software and logistic limitations, and animals were randomly allocated into different groups. All of the studies were sufficiently powered to yield statistically significant results (95% confidence interval). The data were analyzed by one-way or two-way ANOVA, and a P value less than 0.05 was considered significant. Survival curves were plotted using the Kaplan–Meier method, and the differences in survival between groups were assessed by a log-rank test.
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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.).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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
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.
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
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
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). https://doi.org/10.1038/s43018-020-00119-y
Nature Cancer (2020)