Universal immunotherapeutic strategy for hepatocellular carcinoma with exosome vaccines that engage adaptive and innate immune responses

Abstract

Background: Personalized immunotherapy utilizing cancer vaccines tailored to the tumors of individual patients holds promise for tumors with high genetic heterogeneity, potentially enabling eradication of the tumor in its entirety.

Methods: Here, we demonstrate a general strategy for biological nanovaccines that trigger tailored tumor-specific immune responses for hepatocellular carcinoma (HCC). Dendritic cell (DC)-derived exosomes (DEX) are painted with a HCC-targeting peptide (P47-P), an α-fetoprotein epitope (AFP212-A2) and a functional domain of high mobility group nucleosome-binding protein 1 (N1ND-N), an immunoadjuvant for DC recruitment and activation, via an exosomal anchor peptide to form a "trigger" DEX vaccine (DEX_P&A2&N).

Results: DEX_P&A2&N specifically promoted recruitment, accumulation and activation of DCs in mice with orthotopic HCC tumor, resulting in enhanced cross-presentation of tumor neoantigens and de novo T cell response. DEX_P&A2&N elicited significant tumor retardation and tumor-specific immune responses in HCC mice with large tumor burdens. Importantly, tumor eradication was achieved in orthotopic HCC mice when antigenic AFP peptide was replaced with the full-length AFP (A) to form DEX_P&A&N. Supplementation of Fms-related tyrosine kinase 3 ligand greatly augmented the antitumor immunity of DEX_P&A&N by increasing immunological memory against tumor re-challenge in orthotopic HCC mice. Depletion of T cells, cross-presenting DCs and other innate immune cells abrogated the functionality of DEX_P&A&N.

Conclusions: These findings demonstrate the capacity of universal DEX vaccines to induce tumor-specific immune responses by triggering an immune response tailored to the tumors of each individual, thus presenting a generalizable approach for personalized immunotherapy of HCC, by extension of other tumors, without the need to identify tumor antigens.

Keywords: Adaptive and innate immunity; Exosome; Hepatocellular carcinoma; Personalized immunotherapy.

Fig. 1

Evaluation of DEX_P&A2&N’s tumor-targeting, DC-recruiting and DC-activating ability in orthotopic mCherry-expressing HCC mice. a Schematic illustration for designer DEX vaccine-DEX_P&A2&N. P-P47; A2-AFP212; N-N1ND. DEX refers to DC-derived exosomes. b Flow cytometric analysis to assess the simultaneous binding efficiency of three moieties on DEX (n = 4). Diagram for dosing regimen (c) and tissue distribution and quantitative analysis of labeled DEX_P&A2&N (d) in day-14 orthotopic HCC mice bearing mCherry-expressing tumors. DiR-labeled DEX_A2&N (n = 9), DEX_P&A2&N (n = 9) (80 μg/mouse) or PBS (n = 4) were injected into day-14 orthotopic HCC mice bearing mCherry-expressing tumors intravenously, and tissues were harvested 2 h after injection (one-way ANOVA post hoc Student–Newman–Keuls test was used except for liver and tumor in which one-way ANOVA on ranks was used). mLN-mesenteric lymph node; iLN-inguinal lymph node; Tu-tumor. e Flow cytometric and quantitative analysis of CD11c⁺ DCs in tumor-infiltrating lymphocytes (TILs) from HCC mice bearing mCherry-expressing tumors (n = 13; one-way ANOVA post hoc Student–Newman–Keuls test). Flow cytometric (f) and quantitative analysis (g) of CD103⁺CD11c⁺ (one-way ANOVA on ranks) and CD8α⁺ CD11c⁺ DCs (one-way ANOVA post hoc Student–Newman–Keuls test) in TILs from HCC mice (n = 9). h Flow cytometric and quantitative analysis of surface protein markers on CD11c⁺ DCs from tumors of orthotopic HCC mice treated with DEX_P&A2 or DEX_P&A2&N (n = 5; one-way ANOVA post hoc Student–Newman–Keuls test). i Flow cytometric and quantitative analysis of surface protein markers on CD103⁺CD11c⁺ and CD8α⁺CD11c⁺ DCs from tumors of orthotopic HCC mice treated with DEX_P&A2 or DEX_P&A2&N (n = 5; one-way ANOVA post hoc Student–Newman–Keuls test). *p < 0.05, **p < 0.001; n.s, not significant

Fig. 2

Investigation of DEX_P&A2&N promoting DC uptake and cross-presentation of tumor neoantigens in orthotopic HCC mice. a Diagram for dosing regimen of DEX_P&A2&N. DEX_P&A2 or DEX_P&A2&N (80 μg/mouse) were injected into day-14 orthotopic HCC mice bearing mCherry-expressing tumors intravenously, and tissues were harvested 2 days after injection. Flow cytometric (b) and quantitative analysis (c) of mCherry⁺CD11c⁺ DCs from tumor of orthotopic HCC mice treated with DEX_P&A2 or DEX_P&A2&N (n = 11) (One-way ANOVA on ranks was used for the number and one-way ANOVA post hoc Student–Newman–Keuls test was applied for the percent analysis). TiDC: tumor-infiltrating DC. Flow cytometric (d) and quantitative analysis (e) of mCherry⁺CD103⁺CD11c⁺ or CD8α⁺CD11c⁺ DCs from tumor of orthotopic HCC mice treated with DEX_P&A2&N (n = 8; Mann–Whitney rank-sum test). f Diagram for dosing regimen of DEX_P&A2&N. DEX_P&A2 or DEX_P&A2&N (80 μg/mouse) were injected into day-14 orthotopic HCC mice bearing OVA-expressing tumors intravenously, and tissues were harvested 2 days after injection. g Flow cytometric and quantitative analysis of OVA⁺ tetramer T cells from splenocytes of orthotopic HCC mice treated with DEX_P&A2 or DEX_P&A2&N (n = 5; one-way ANOVA on ranks). Flow cytometric (h) and quantitative analysis (i) of CD103⁺CD11c⁺ or CD8α⁺CD11c⁺ DCs from TILs of orthotopic Batf3^−/− HCC mice treated with PBS or DEX_P&A2 (n = 4) or DEX_P&A2&N (n = 5) and orthotopic wild-type (WT) HCC mice treated with DEX_P&A2&N (n = 5) (one-way ANOVA on ranks). j Flow cytometric and quantitative analysis of OVA⁺ tetramer T cells from tumor of orthotopic Batf3^−/− HCC mice treated with PBS or DEX_P&A2 (n = 4) or DEX_P&A2&N (n = 5) and orthotopic wild-type (WT) HCC mice treated with DEX_P&A2&N (n = 5) (one-way ANOVA post hoc Student–Newman–Keuls test). *p < 0.05, **p < 0.001; n.s, not significant

Fig. 3

Antitumor effects of DEX_P&A2&N in orthotopic HCC mice. a Diagram for dosing regimen of DEX_P&A2&N. DEX_P&A2 or DEX_P&A2&N (80 μg/mouse) were injected into day-7 orthotopic HCC mice intravenously three times weekly, and tissues were harvested 2 days after last injection. b Measurement of tumor volume in day-7 orthotopic HCC mice treated with DEX_P&A2 or DEX_P&A2&N (n = 10; one-way ANOVA on ranks). c Flow cytometric and quantitative analysis of AFP⁺ tetramer T cells from splenocytes of orthotopic HCC mice treated with DEX_P&A2 or DEX_P&A2&N (n = 4; one-way ANOVA post hoc Student–Newman–Keuls test). d Flow cytometric and quantitative analysis of GPC3⁺ tetramer T cells from splenocytes of orthotopic HCC mice treated with DEX_P&A2 or DEX_P&A2&N (n = 5; one-way ANOVA post hoc Student–Newman–Keuls test). Flow cytometric and quantitative analysis of IFN-γ⁺CD8⁺ T cells (e) (n = 5; one-way ANOVA post hoc Student–Newman–Keuls test) and T cell division (f) (n = 9; one-way ANOVA on ranks) in splenocytes of orthotopic HCC mice treated with DEX_P&A2 or DEX_P&A2&N, followed by in vitro stimulation of GPC3 epitopes. g Diagram for dosing regimen of DEX_P&A2&N. DEX_P&A2 or DEX_P&A2&N (120 μg/mouse) were injected into day-21 orthotopic HCC mice bearing large established tumors intravenously three times weekly, and tissues were harvested 2 weeks after last injection. h MRI monitoring of tumor growth in day-21 orthotopic HCC mice bearing large established tumors at different time points. i Assessment of tumor size in orthotopic HCC mice bearing large established tumors treated with PBS (n = 16), DEX_P&A2 (n = 7) or DEX_P&A2&N (n = 16) at 28 days after initial treatment (one-way ANOVA on ranks). j Survival rate of orthotopic HCC mice treated with PBS (n = 9), DEX_P&A2 or DEX_P&A2&N (n = 8). *p < 0.05, **p < 0.001; n.s, not significant

Fig. 4

Antitumor immunity of DEX_P&A&N in orthotopic HCC mice. a Flow cytometric analysis to assess the double loading efficiency of two moieties on DEX_AFP. DEX_P&A&N refer to DEX_P47&AFP&N1ND. b Diagram for dosing regimen of DEX_P&A&N. DEX_AFP or DEX_P&A&N (80 μg/mouse) were injected into day-7 orthotopic HCC mice intravenously three times weekly, and tissues were harvested 7 days after last injection. c Dynamic monitoring of tumor growth in orthotopic HCC mice with MRI at different time points. d Measurement of tumor size in orthotopic HCC mice at 21 days after priming (n = 20; one-way ANOVA on ranks). e Flow cytometric and quantitative analysis of GPC3⁺ tetramer T cells from tumor of orthotopic HCC mice treated with DEX_AFP or DEX_P&A&N (n = 5; one-way ANOVA post hoc Student–Newman–Keuls test). f Diagram for dosing regimen of DEX_P&A&N. DEX_AFP or DEX_P&A&N (120 μg/mouse) were injected into day-21 orthotopic HCC mice bearing large established tumors intravenously three times weekly, and tissues were harvested 2 weeks after last injection. g Dynamic monitoring of tumor growth in orthotopic HCC mice bearing large established tumors treated with DEX_AFP or DEX_P&A&N with MRI at different time points. h Measurement of tumor size in orthotopic HCC mice at 28 days after priming (n = 5; one-way ANOVA post hoc Student–Newman–Keuls test). i Flow cytometric and quantitative analysis of AFP⁺ tetramer T cells from tumor of orthotopic HCC mice treated with DEX_AFP or DEX_P&A&N (n = 5; one-way ANOVA post hoc Student–Newman–Keuls test). *p < 0.05, **p < 0.001; n.s, not significant

Fig. 5

DEX_P&A&N evoke potent antitumor immunity with efficacy dependent on innate and adaptive immune responses. Flow cytometric and quantitative analysis of tumor-infiltrating CD8⁺ (a) and ratio of tumor-infiltrating CD8⁺ to CD4⁺ (b) and flow cytometric and quantitative analysis of tumor CD4⁺CD25⁺ (c) T cells of DEX_P&A&N -treated orthotopic HCC mice bearing large established tumors (n = 5). d  Diagram for dosing regimen of DEX_P&A&N. DEX_AFP or DEX_P&A&N (80 μg) were injected into day-7 thymus-deficient nude mice bearing subcutaneous HCC tumors intravenously three times weekly, and tissues were harvested 2 days after last injection. e Measurement of tumor size in nude mice bearing subcutaneous HCC tumors at different time points (n = 5). One-way ANOVA on ranks test was used for day 7 and 21; one-way ANOVA post hoc Student–Newman–Keuls test was used for day 9, 11, 14, 16, 18 and 23. f Flow cytometric and quantitative analysis of tumor-infiltrating CD8⁺ T cells of DEX_P&A&N-treated orthotopic Batf3^−/− HCC mice (n = 5; one-way ANOVA on ranks). g Western blot to examine Hepa1-6 tumor cell lysates with serum from DEX_P&A&N- or DEX_AFP-treated mice. α-actin was used as a loading control. h Representative images and quantification of tumor nodules in lungs 21 days after serum re-infusion and tumor challenge (n = 4). i Flow cytometric and quantitative analysis of tumor-infiltrating NK cells of DEX_P&A&N-treated orthotopic HCC mice bearing large established tumors (n = 5). j Diagram for NK depletion experiments. k Measurement of tumor volume in DEX_P&A&N and anti-NK1.1-treated ectopic HCC mice at different time points (n = 5). One-way ANOVA post hoc Student–Newman–Keuls test was used for day 7, 9, 11, 14, 21 and 23; one-way ANOVA on ranks test was applied for day 16 and 18; and Two-tailed t test was applied for DEX_P&A&N with or without anti-NK1.1 antibody on day 23. *p < 0.05, **p < 0.001; n.s means not significant. Note: One-way ANOVA post hoc Student–Newman–Keuls test was used for Fig. 5a-c and 5 h, 5i

Fig. 6

Flt3L augments DEX_P&A&N’s antitumor potency in orthotopic HCC mice. a Diagram for dosing regimen of DEX_P&A&N and Flt3L in orthotopic HCC mice. Flt3L was administered subcutaneously to day-3 orthotopic HCC mice at the dose of 800 μg /kg/day for 8 days consecutively and DEX_P&A&N (80 μg/mouse) were intravenously administered on day 7 after tumor implantation three times weekly. b Dynamic monitoring of tumor growth in orthotopic HCC mice treated with DEX_P&A&N or DEX_P&A&N and Flt3L with MRI at different time points. c Measurement of tumor volume for each individual mouse treated with PBS, DEX_P&A&N or DEX_P&A&N and Flt3L (n = 6). One-way ANOVA on ranks test was used for day 21, 28 and 42; one-way ANOVA post hoc Student–Newman–Keuls test was used for day 14. TF means tumor-free. d Quantitative analysis of CD8⁺ T cells and ratio of CD8⁺ to CD4⁺ T cells in blood of orthotopic HCC mice treated with PBS (n = 4), DEX_P&A&N or DEX_P&A&N and Flt3L (n = 6) at day 42 after tumor implantation (One-way ANOVA on ranks). Flow cytometric and quantitative analysis of AFP⁺ tetramer (e) and GPC3⁺ tetramer (f) T cells in blood of orthotopic HCC mice treated with PBS (n = 4), DEX_P&A&N or DEX_P&A&N and Flt3L (n = 6) at day 42 after tumor implantation (one-way ANOVA post hoc Student–Newman–Keuls test). g Assessment of IFN-γ and TGF-β in blood of orthotopic HCC mice treated with PBS (n = 4), DEX_P&A&N or DEX_P&A&N and Flt3L (n = 6) at day 42 after tumor implantation (one-way ANOVA post hoc Student–Newman–Keuls test). *p < 0.05, **p < 0.001; n.s, not significant

Fig. 7

Long-term protective memory immune responses elicited by DEX_P&A&N and Flt3L against tumor re-challenge. Flow cytometric and quantitative analysis of CD44^hi CD8⁺ (a) and CD62L^lowCD44^hiCD8⁺ (b) T cells in blood of orthotopic HCC mice treated with PBS (n = 4), DEX_P&A&N, DEX_P&A&N and Flt3L (n = 6) at day 42 after tumor implantation (one-way ANOVA post hoc Student–Newman–Keuls test). c Diagram for tumor re-challenge in DEX_P&A&N and Flt3L-treated tumor-free mice. d Measurement of tumor volume in DEX_P&A&N and Flt3L-treated tumor-free mice after re-challenge (n = 4). Mann–Whitney rank-sum test was used for day 7; one-way ANOVA on ranks test was used for day 14, 21, 28, 35 and 42; one-way ANOVA post hoc Student–Newman–Keuls test was used for day 49. e Survival rate of DEX_P&A&N and Flt3L-treated tumor-free mice after re-challenge (n = 4). Flow cytometric and quantitative analysis of CD44^hiCD8⁺ (f) and CD62L^lowCD44^hi CD8⁺ (g) T cells in blood of DEX_P&A&N and Flt3L-treated tumor-free mice at different time points after tumor re-challenge (n = 4; one-way ANOVA post hoc Student–Newman–Keuls test). *p < 0.05, **p < 0.001; n.s, not significant

Fig. 8

Graphical abstract for illustrating the mechanism underlying the antitumor immunogenicity of universal DEX vaccines