STING-activating dendritic cell-targeted nanovaccines that evoke potent antigen cross-presentation for cancer immunotherapy

Abstract

Recently, nanovaccine-based immunotherapy has been robustly investigated due to its potential in governing the immune response and generating long-term protective immunity. However, the presentation of a tumor peptide-major histocompatibility complex to T lymphocytes is still a challenge that needs to be addressed for eliciting potent antitumor immunity. Type 1 conventional dendritic cell (cDC1) subset is of particular interest due to its pivotal contribution in the cross-presentation of exogenous antigens to CD8⁺ T cells. Here, the DC-derived nanovaccine (denoted as Si9GM) selectively targets cDC1s with marginal loss of premature antigen release for effective stimulator of interferon genes (STING)-mediated antigen cross-presentation. Bone marrow dendritic cell (BMDC)-derived membranes, conjugated to cDC1-specific antibody (αCLEC9A) and binding to tumor peptide (OVA_257-264), are coated onto dendrimer-like polyethylenimine (PEI)-grafted silica nanoparticles. Distinct molecular weight-cargos (αCLEC9A-OVA_257-264 conjugates and 2'3'-cGAMP STING agonists) are loaded in hierarchical center-radial pores that enables lysosome escape for potent antigen-cross presentation and activates interferon type I, respectively. Impressively, Si9GM vaccination leads to the upregulation of cytotoxic T cells, a reduction in tumor regulatory T cells (Tregs), M1/M2 macrophage polarization, and immune response that synergizes with αPD-1 immune checkpoint blockade. This nanovaccine fulfills a dual role for both direct T cell activation as an artificial antigen-presenting cell and DC subset maturation, indicating its utility in clinical therapy and precision medicine.

Keywords: Antigen cross-presentation; Artificial antigen-presenting cells; DC-based nanovaccines; STING pathway activation; Type 1 conventional dendritic cells.

Graphical abstract

Scheme 1

Fabrication and therapeutic mechanisms of Si9GM. Fabrication steps: bone mesenchymal stem cells were isolated from femurs and tibias of C57BL/6 mice, and then stimulated to obtain immature bone marrow-derived dendritic cells (BMDCs). After activation, mature BMDCs were collected, followed by extraction to obtain BMDC membranes (BMDCm). Next, antigen peptides (OVA_257-264) were loaded onto BMDCm by binding to major histocompatibility complex class I (MHC I) molecules using a pH-dependent method. The obtained membranes (DC-OVA_257-264) were then conjugated with αCLEC9A using sulfo-SMCC/Traut's reagent linkers to produce BMDC membranes containing OVA_257-264 and αCLEC9A antibodies, denoted as αCLEC9A@DC-OVA_257-264. In addition, dendrimer-like hierarchical large pore silica nanoparticles were synthesized using the quasi-emulsion nanodroplet method. These nanoparticles were then loaded with αCLEC9A-OVA_257-264 conjugates and 2′3′-cGAMP STING agonist, denoted as Si9G. Finally, Si9G NPs were coated with αCLEC9A@DC-OVA_257-264 to generate Si9GM nanovaccines. Therapeutic mechanisms of Si9GM: Si9GM nanovaccines were effectively delivered to LNs, where Si9GM acts both as an artificial antigen-presenting cell and as a DC activator for directly presenting antigen on CD8⁺ T cells and DC maturation, respectively. αCLEC9A on the Si9GM surface selectively target type 1 conventional DCs (cDC1s), where the released αCLEC9A-OVA_257-264 conjugates and 2′3′-cGAMP STING agonist play critical roles in antigen cross-presentation and STING pathway activation, respectively. Furthermore, nanovaccine Si9GM in combination with immune checkpoint blockade (αPD-1) promoted potent antitumor immunity for effective cancer immunotherapy and metastasis inhibition.

Fig. 1

Preparation and characterization of Si9GM. (A) Schematic representation of Si9GM nanovaccine formulation involving coating modified BMDC membranes on center-radial large pore silica NPs, loading αCLEC9A-OVA_257-264 conjugates and 2′3′-cGAMP STING agonists. (B) TEM and SEM images of DHPSi NPs and Si9GM (white scale bars: 50 nm), yellow arrows: projections of BMDC membranes. (C) Hydrodynamic size of DHPSi-NH₂, BMDCm, and Si9GM. (D) Zeta potential of DHPSi-NH₂, DHPSi-COOH, and DHPSi-PEI. (E) SDS-PAGE of BMDC lysates and BMDC membranes extracted from BMDCs post LPS-induced activation (1 μg mL⁻¹) for 24 h (denoted as BMDCm). (F) Western blot analysis of surface proteins of cell lysates and BMDCm and antigen peptide loaded BMDCm (denoted as DC-OVA_257-264). (G) Western blot assays of membrane-derived biomarkers and intracellular proteins in the BMDC lysate and BMDCm-extracted protein. (H) Flow cytometric analysis of the presence of antigen peptides OVA_257-264 on BMDC membranes after loading antigen fragments on MHC I molecules by pH-dependent mechanism, blue: unstained BMDCm, pink: DC-OVA_257-264 (OVA_257-264: 50 μg per 1 mg of BMDCm), green: DC-OVA_257-264 (OVA_257-264: 100 μg per 1 mg of BMDCm). (I) Analysis of the loading of antigen peptides OVA_257-264 on BMDCm by UV–vis measurement. (J) Analysis of the presence of MHC I on BMDCm by flow cytometry, blue: unstained BMDCm, grey: 1-month stored BMDCm, red: 2-month stored BMDCm. (K) TEM image DC-OVA_257-264 (yellow arrows: dendrite structure of DC). (L) Energy dispersive spectroscopy (EDS) analysis of Au-labeled antibody-conjugated αCLEC9A@ DC-OVA_257-264, showing the presence of Au atoms represented for the successful conjugation of αCLEC9A on the surface of DC-OVA_257-264. Scale bar: 100 nm. (M) Zeta potential of DHPSi NPs loading αCLEC9A-OVA_257-264 conjugates and 2′3′-cGAMP agonists (denoted as Si9G), BMDCm, and DC-OVA_257-264 coated Si9G nanovaccine (denoted as Si9GM). (N) EDS mapping analysis of elements (Si, O, N, P, S) in Si9GM nanovaccine.

Fig. 2

DC internalization of Si9GM. (A) Bio-TEM images of Si9GM internalized into BMDCs, the yellow arrows point out the uptake of Si9GM, scale bars: 5 μm and 1 μm. (B) Cellular uptake of Si9GM into BMDCs was analyzed by CLSM, green: neuro-DiO; red: DiI, blue: Hoechst 33342, scale bar: 20 μm. (C) Determination of αCLEC9A-OVA_257-264 and 2′3′-cGAMP delivered by Si9GM into cDC1s by CLSM, blue: nucleus; red: AF647-labeled CLEC9A conjugate; green: cFAET-labeled 2′3′cGAMP, scale bar: 10 μm. (D) Investigation of lysosome escape ability of Si9GM in cDC1s, blue: nucleus; red: lysotracker Red; green: Si9GM loading cFAET-labeled 2′3′-cGAMP, scale bar: 10 μm. (E) Analysis of free 2′3′-cGAMP uptake into cDC1s by flow cytometry. (F) Analysis of 2′3′-cGAMP delivered by Si9GM into cDC1s by flow cytometry. (G) Comparison of the uptake of free αCLEC9A conjugates and αCLEC9A-OVA_257-264 conjugate delivered by Si9GM into cDC1s by flow cytometry. Column graphs showed data presented as the mean ± standard deviation, with statistical significance calculated via two-tailed Student's t-test (F,G). P > 0.05 stands for not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3

(A) Evaluation of the role of Si9GM as an artificial antigen-presenting cell through the interaction with CD8⁺ T cell, blue: Hoechst 33342, green: neuro-DiO, red: DiI, scale bar: 5 μm). (B, C) Representative flow cytometric plots and percentage of activated CD8⁺ OT-I T cells after 20 h of various treatments. (D, E) Representative flow cytometric plots and percentage of proliferating CD8⁺ OT-I T cells after 4 days of various treatments. (F, G) Representative flow cytometric plots and viability percentage of cytotoxic CD8⁺ T cell-induced cancer cells after 20-h co-incubation. The data is presented as the mean ± standard deviation, with statistical significance determined using one-way ANOVA with Tukey's test (C, E, G) with Tukey's test (F). P > 0.05 stands for not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4

STING pathway activation and biodistribution of Si9GM for DC maturation and T cell interaction. (A) Schematic illustration of STING pathway activation. (B) Western blot assay of 2′3′-cGAMP, and Si9GM inducing TBK1, STING, NF-κB and IRF3 phosphorylation in BMDCs in vitro. (C) ELISA assay of IFN-β produced by 2′3′-cGAMP, and Si9GM in BMDCs in vitro. (D) Flow cytometric analysis of surface biomarkers for DC maturation, antigen cross-presentation, and LN homing ability of Si9GM, free 2′3′-cGAMP in BMDCs. (E) Biodistribution of Si9GM and free 2′3′-cGAMP in C57BL/6 mice within 48 h post subcutaneous injection. (F) Relative fluorescent intensity of inguinal LNs in the Si9GM-treated group with elapsing time post subcutaneous injection. (G) Ex vivo fluorescence imaging of cFAET-labeled 2′3′-cGAMP and cFAET-labeled 2′3′cGAMP@Si9GM distributions in the inguinal LNs and major organs (heart, lung, spleen, liver and kidneys) at 12 h post vaccination. (H) Fluorescent intensity of excised inguinal LNs and main organs (heart, lung, spleen, liver and kidneys) at 12 h after vaccination. (I) Fluorescence images of DiR-labeled@Si9GM sample, excised C57BL/6 mouse at 12 h post-vaccination, excised inguinal LNs, and major organs to analyze LN homing ability of Si9GM originated from the injection site. (J) Immunofluorescent staining of LNs from mice vaccinated with Si9GM, green: CD3⁺ T cells, red: Si9GM nanovaccine, blue: nuclei, and intensity profile of fluorescence at a specific site in the CLSM image of inguinal LNs; scale bar: (i) 500 μm, (ii) 100 μm. The data is presented as the mean ± standard deviation, with statistical significance determined using one-way ANOVA with Tukey's test (D) or two-way ANOVA with Tukey's test (F). P > 0.05 stands for not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5

Si9GMs augment the maturation of cDC1s and CD8⁺ T-cell priming. (A) Schematic illustration of in vivo experiments to analyze the STING pathway activation in cDC1s triggering CD8⁺ T-cell priming. The 2′3′-cGAMP and Si9GM were administered by s.c. injection, and DCs and T cells were collected at day 8 and day 10 post the first injection, respectively for flow cytometric analysis. (B) Quantitative evaluation of immune cells (CD45⁺ cells) and DC population (CD11c⁺ MHC II⁺) from different treatments. (C) Representative flow cytometric plots of the percentage of cDC1 subsets in DCs: CD8α cDC1 (gated on CD45⁺ CD11c⁺ MHC II⁺) and CD103⁺ cDC1 (gated on CD45⁺ CD11c⁺ MHC II⁺). (D) Illustrative flow cytometric graphs of costimulatory molecule CD86 and quantification of CD86 percentage in each cDC1 subset. (E) Representative flow cytometric graphs and percentage of antigen SIINFEKL-presented DCs in response to various treatments. (F) Representative flow cytometric graphs and populations of OVA-specific CD8⁺ T cells. (G) The population of IFN-γ⁺ CD8⁺ cells in tumors collected from mice treated with 2′3′-cGAMP and Si9GM compared to the untreated group. The data is presented as the mean ± standard deviation, with statistical significance calculated using one-way ANOVA with Tukey's test (B,C,D) or one-way ANOVA with Tukey's test (E,F). P > 0.05 stands for not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 6

In vivo study of B16-OVA tumor inhibition. (A) Schematic illustration of treatment schedule for melanoma cancer in C57BL/6 mice. (B) Scheme illustrating the role of various immune cells involved in antitumor immunity. (C) Graph presenting tumor volume growth of the B16-OVA tumor-bearing C57BL-6 mice following different treatments over a 16-day period (n = 5). (D) Individual curves of B16-OVA tumor growth of the mice over a 16-day period (n = 5). (E) Excised tumor weight of B16-OVA tumor-bearing mice after 16 days of treatment. (F) Illustrative photos of tumors in each group at the end of treatment. (G) Representative flow cytometric graphs and proportions of mature DCs (CD11c⁺ CD80⁺ CD86⁺) in spleens at the end of the schedule. (H) Illustrative flow cytometric graphs and proportions of T cells in tumor tissues observed on day 7 after the last vaccination. (I) The proportions of M1-like, M2-like macrophages in TAMs and the M1/M2 ratio in tumor tissues observed on day 7 after the last vaccination. (J) Illustrative flow cytometric graphs and percentages of regulatory CD4⁺ T cells in spleens. (K) Illustrative flow cytometric graphs and proportions of SIINFEKL⁺ CD8⁺ T cells in tumors. (L) Illustrative flow cytometric graphs and percentages of activated T cells (CD3⁺ CD8⁺ CD69⁺) in LNs at day 3 after the last vaccination. (M) Illustrative flow cytometric graphs and proportions of SIINFEKL⁺ CD11c⁺ DC cells in spleens at day 3 after the last vaccination. (N) Illustrative flow cytometric graphs and proportions of tumor-infiltrated IFNγ⁺ NK1.1⁺ NK cells. (O) Representative flow cytometric graphs and percentages of tumor-infiltrated IFNγ⁺ CD8⁺ T cells in tumors on day 7 after the last vaccination. (P) Illustrative flow cytometric graphs and percentages of tumor-infiltrated GrnB⁺ CD8⁺ T cells in tumors on day 7 after the last vaccination. (Q) Illustrative flow cytometric graphs and proportions of CD8⁺ T cells, memory effector T cells, and memory central T cells in tumors. (R) Immunofluorescent images of tumors stained by CD8 antibody, DAPI. (S) Immunofluorescent images of tumors stained by Ki67 antibody, DAPI. (T) ELISA assay of inflammatory cytokine levels (IFN-γ, TNF-α, IL-2, IL-6, and IFN-β) in serum collected from B16-OVA tumor-bearing mice at 72 h post final vaccination. The data is presented as the mean ± standard deviation, with statistical significance determined using one-way ANOVA with Tukey's test. P > 0.05 stands for not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 6

In vivo study of B16-OVA tumor inhibition. (A) Schematic illustration of treatment schedule for melanoma cancer in C57BL/6 mice. (B) Scheme illustrating the role of various immune cells involved in antitumor immunity. (C) Graph presenting tumor volume growth of the B16-OVA tumor-bearing C57BL-6 mice following different treatments over a 16-day period (n = 5). (D) Individual curves of B16-OVA tumor growth of the mice over a 16-day period (n = 5). (E) Excised tumor weight of B16-OVA tumor-bearing mice after 16 days of treatment. (F) Illustrative photos of tumors in each group at the end of treatment. (G) Representative flow cytometric graphs and proportions of mature DCs (CD11c⁺ CD80⁺ CD86⁺) in spleens at the end of the schedule. (H) Illustrative flow cytometric graphs and proportions of T cells in tumor tissues observed on day 7 after the last vaccination. (I) The proportions of M1-like, M2-like macrophages in TAMs and the M1/M2 ratio in tumor tissues observed on day 7 after the last vaccination. (J) Illustrative flow cytometric graphs and percentages of regulatory CD4⁺ T cells in spleens. (K) Illustrative flow cytometric graphs and proportions of SIINFEKL⁺ CD8⁺ T cells in tumors. (L) Illustrative flow cytometric graphs and percentages of activated T cells (CD3⁺ CD8⁺ CD69⁺) in LNs at day 3 after the last vaccination. (M) Illustrative flow cytometric graphs and proportions of SIINFEKL⁺ CD11c⁺ DC cells in spleens at day 3 after the last vaccination. (N) Illustrative flow cytometric graphs and proportions of tumor-infiltrated IFNγ⁺ NK1.1⁺ NK cells. (O) Representative flow cytometric graphs and percentages of tumor-infiltrated IFNγ⁺ CD8⁺ T cells in tumors on day 7 after the last vaccination. (P) Illustrative flow cytometric graphs and percentages of tumor-infiltrated GrnB⁺ CD8⁺ T cells in tumors on day 7 after the last vaccination. (Q) Illustrative flow cytometric graphs and proportions of CD8⁺ T cells, memory effector T cells, and memory central T cells in tumors. (R) Immunofluorescent images of tumors stained by CD8 antibody, DAPI. (S) Immunofluorescent images of tumors stained by Ki67 antibody, DAPI. (T) ELISA assay of inflammatory cytokine levels (IFN-γ, TNF-α, IL-2, IL-6, and IFN-β) in serum collected from B16-OVA tumor-bearing mice at 72 h post final vaccination. The data is presented as the mean ± standard deviation, with statistical significance determined using one-way ANOVA with Tukey's test. P > 0.05 stands for not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 7

Inhibition of lung metastasis of Si9GM combined with αPD-1 ICB in melanoma cancer. (A) Schematic illustration of lung metastasis inhibition treatment. (B) Excised lungs in all groups at the end of treatment. (C) Weight of excised lungs at the end of treatment. (D) H & E staining of excised lungs. E, F) Illustrative flow cytometric plots and proportions of IFNγ⁺ NK1.1⁺ cells in lungs. All data are presented as the mean ± standard deviation, with statistical significance calculated via one-way ANOVA with Tukey's test. P > 0.05 stands for not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 8

Investigation of the role of immune cells in lung metastasis prevention. (A) Schematic overview of lung metastasis inhibition in the depletion of NK cells (G3) and CD8 T cells (G4) compared to the untreated group (G1), and IgG2a isotype injection (G2). (B) Excised lungs at the end of treatment. (C) H & E staining of excised lungs in G1, G2, G3, and G4. (D) Weight of excised lungs in G1, G2, G3 and G4 group. (E) Analysis of the population of cDC1s subsets (CD8α⁺ cDC1), (CD103⁺ cDC1) in lungs (gated on CD45⁺ CD11c⁺ MHCII⁺) by flow cytometry. (F) Illustrative flow cytometric graphs and proportions of IFNγ⁺ NK1.1⁺ cells in lungs by flow cytometric evaluation. (G) Examination of T cell population in lungs at the end of treatment. Data are presented as the mean ± standard deviation, with statistical significance calculated via one-way ANOVA with Tukey's test (D), or two-way ANOVA with Tukey's test (E,G), or two-tailed Student's t-test (F). P > 0.05 stands for not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.