Efficient Lymph Node-Targeted Delivery of Personalized Cancer Vaccines with Reactive Oxygen Species-Inducing Reduced Graphene Oxide Nanosheets

Abstract

Therapeutic cancer vaccines require robust cellular immunity for the efficient killing of tumor cells, and recent advances in neoantigen discovery may provide safe and promising targets for cancer vaccines. However, elicitation of T cells with strong antitumor efficacy requires intricate multistep processes that have been difficult to attain with traditional vaccination approaches. Here, a multifunctional nanovaccine platform has been developed for direct delivery of neoantigens and adjuvants to lymph nodes (LNs) and highly efficient induction of neoantigen-specific T cell responses. A PEGylated reduced graphene oxide nanosheet (RGO-PEG, 20-30 nm in diameter) is a highly modular and biodegradable platform for facile preparation of neoantigen vaccines within 2 h. RGO-PEG exhibits rapid, efficient (15-20% ID/g), and sustained (up to 72 h) accumulation in LNs, achieving >100-fold improvement in LN-targeted delivery, compared with soluble vaccines. Moreover, RGO-PEG induces intracellular reactive oxygen species in dendritic cells, guiding antigen processing and presentation to T cells. Importantly, a single injection of RGO-PEG vaccine elicits potent neoantigen-specific T cell responses lasting up to 30 days and eradicates established MC-38 colon carcinoma. Further combination with anti-PD-1 therapy achieved great therapeutic improvements against B16F10 melanoma. RGO-PEG may serve a powerful delivery platform for personalized cancer vaccination.

Keywords: cancer immunotherapy; cancer vaccine; nanosheet; positron emission tomography; reduced graphene oxide.

Figure 1.

RGO-PEG nanoplatform for cancer vaccination. (a) Schematic illustration of RGO(CpG)-PEG-neoantigen for LN-targeted delivery of antigens and adjuvants. RGO-PEG nanoplatform coloaded with neoantigen peptides and CpG is administered subcutaneously, leading to efficient delivery to APCs in local LNs, generation of intracellular ROS, and elicitation of robust antitumor T cell immunity. (b, c) AFM images and height analysis (inset) of RGO-PEG. (d) Hydrodynamic size analysis of RGO-PEG and RGO(CpG)-PEG-Adpgk. (e) UV–vis absorption spectrum of GO, RGO-PEG, and RGO(CpG)-PEG-Adpgk. (f) Release profile of Adpgk and CpG from RGO(CpG)-PEG-Adpgk kept in PBS at RT. (g) Raman spectra of RGO-PEG, RGO-PEG incubated with H₂O₂ (50 mM), and RGO-PEG incubated with human myeloperoxidase (hMPO, 1 mg/mL) + H₂O₂ (50 mM) at 37 °C for 72 h.

Figure 2.

RGO-PEG promotes intracellular ROS, antigen presentation on DCs, and induction of CD8α⁺ T cells. (a) CD40, CD80, and CD86 expression levels on BMDCs and (b) TNF-α and IL-12(p70) secretion by BMDCs incubated with PBS, RGO-PEG, soluble CpG, or RGO(CpG)-PEG for 8 h. (c) Confocal microscopy images of BMDCs incubated with RGO-PEG-Cy5 (red). BMDCs were stained with Lysotracker dye. (d) LysoSensor fluorescence intensity and (e) intracellular DCF; (f) DQ-OVA fluorescence intensity in BMDCs incubated with the indicated formulations with or without ROS inhibitor NAC (5 mM) for 24 h (d, f) or 4 h (e). (g) SIINFEKL-H-2K^b presentation was quantified on BMDCs after incubation with the indicated formulations for 24 h. *, #, and & indicate statistical differences between group 4 and group 2 (*), 5 (&), or 6 (#). (h) CFSE dilution of OT-I CD8α⁺ T cells after 48 h of coculture with BMDCs pretreated as in (g). Data represent mean ± SEM from a representative experiment (n = 3) from three independent experiments (a–e) or two independent experiments (f–h). Data were analyzed by one-way ANOVA (a, b, d–f, h) or two-way ANOVA (g) with Tukey’s HSD multiple comparison post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3.

RGO-PEG promotes efficient antigen delivery to APCs in LNs. (a) Serial PET images of C57BL/6 mice after SC administration of ⁶⁴Cu-NOTA-Adpgk + CpG and ⁶⁴Cu-NOTA-RGO(CpG)-PEG-Adpgk. Inguinal and axillary LNs and injection site are indicated by red arrowheads. (b) Time–radioactivity curves of injection site, inguinal and axillary LNs, kidney, intestine, liver, blood, and muscle. (c) ⁶⁴Cu radioactivity in major organs at 72 h postinjection. (d) Cellular uptake of Adpgk and RGO(CpG)-PEG-Adpgk in CD45⁺CD11c⁺ DCs and CD45⁺F4/80⁺ macrophages in inguinal LNs at 24 h postinjection. Data represent mean ± SEM from a representative experiment (n = 4) from two independent experiments (a–d). Data were analyzed by one-way ANOVA (b, d) or two-way ANOVA (c) with Tukey’s HSD multiple comparison post hoc test. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 4.

RGO-PEG exerts potent antitumor efficacy against MC-38 colon carcinoma. (a–c) Prophylactic vaccine study. (a) Study design. C57BL/6 mice were vaccinated SC at the tail base with (1) PBS control; (2) RGO(CpG)-PEG; (3) RGO-PEG-Adpgk; (4) soluble CpG + Adpgk; and (5) RGO(CpG)-PEG-Adpgk, followed by MC-38 tumor cell challenge SC in the flank on day 40. Adpgk peptide and CpG doses were both 15 μg in all treatment groups. (b) Adpgk-specific CD8α⁺ T cells among PBMCs. (c) Tumor growth was measured over time and (d) overall survival curve. (e–h) Therapeutic vaccine study. (e) Study design. MC-38 tumor-bearing mice were vaccinated on day 7 as in (a). (f) Adpgk-specific CD8α⁺ T cells among PBMCs. (g) Tumor growth was measured over time and (h) overall survival curve. (i–p) MC-38 tumor-bearing mice were treated as in (i), and (j) IFN-γ ELISPOT assay was performed on splenocytes ex vivo restimulated with Adpgk peptide on day 20. In parallel, inguinal LNs (k, l) and tumor tissues (m–p) were analyzed for activated CD45⁺CD11c⁺CD86⁺ DCs (k, m), Adpgk-specific CD8α⁺ T cells (l, p), CD3⁺CD8α⁺ T cells (n), and CD3⁺CD4⁺ T cells (o) by flow cytometric analyses. Data represent mean ± SEM from a representative experiment (n = 5, a–h) or (n = 4, i–p) from three independent experiments (a–h) or two independent experiments (i–p). Data were analyzed by one-way ANOVA (j–p) or two-way ANOVA (b, c, f, g) with Tukey’s HSD multiple comparison post hoc test or log-rank (Mantel-Cox) test (d, h). * in (b) and (f) indicates statistical difference between groups 4 and 5. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5.

RGO-PEG for combination immunotherapy against B16F10 melanoma. (a, b) C57BL/6 mice were inoculated with B16F10 cells in the SC flank on day 0 and vaccinated on day 7 in the SC tail base with (1) PBS control; (2) RGO(CpG)-PEG; (3) RGO-PEG-(M27+M30); (4) soluble CpG + (M27+M30); or (5) RGO(CpG)-PEG-(M27+M30). Each dose of M27, M30, and CpG was 15 μg in all treatment groups. (b) Average B16F10 tumor growth. (c–j) B16F10 tumor-bearing C57BL/6 mice were vaccinated on days 7 and 14 with the indicated groups with or without 100 μg of anti-PD-1 IgG treatment on days 10, 13, 16, and 19. (d) Average tumor growth; (e) survival curves; and (f) body weight. IFN-γ ELISPOT assay was performed on day 20 with (g) PBMCs or (h) splenocytes ex vivo restimulated with M27 and M30 peptides. On day 20, tumor-infiltrating CD8α⁺ T cells (i) and activated CD45⁺CD11c⁺CD86⁺ DCs (j) were quantified by flow cytometric analyses. Data represent mean ± SEM from a representative experiment (n = 5, a–f) or (n = 4, g–j) from three independent experiments (a–f) or two independent experiments (g–j). Data were analyzed by one-way ANOVA (g–j) or two-way ANOVA (b, d) with Tukey’s HSD multiple comparison post hoc test or log-rank (Mantel–Cox) test (e). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.