A STING-activating nanovaccine for cancer immunotherapy

Abstract

The generation of tumour-specific T cells is critically important for cancer immunotherapy. A major challenge in achieving a robust T-cell response is the spatiotemporal orchestration of antigen cross-presentation in antigen-presenting cells with innate stimulation. Here, we report a minimalist nanovaccine, comprising a simple physical mixture of an antigen and a synthetic polymeric nanoparticle, PC7A NP, which generates a strong cytotoxic T-cell response with low systemic cytokine expression. Mechanistically, the PC7A NP achieves efficient cytosolic delivery of tumour antigens to antigen-presenting cells in draining lymph nodes, leading to increased surface presentation while simultaneously activating type I interferon-stimulated genes. This effect is dependent on stimulator of interferon genes (STING), but not the Toll-like receptor or the mitochondrial antiviral-signalling protein (MAVS) pathway. The nanovaccine led to potent tumour growth inhibition in melanoma, colon cancer and human papilloma virus-E6/E7 tumour models. The combination of the PC7A nanovaccine and an anti-PD-1 antibody showed great synergy, with 100% survival over 60 days in a TC-1 tumour model. Rechallenging of these tumour-free animals with TC-1 cells led to complete inhibition of tumour growth, suggesting the generation of long-term antitumour memory. The STING-activating nanovaccine offers a simple, safe and robust strategy in boosting anti-tumour immunity for cancer immunotherapy.

Figure 1. PC7A NP induces robust antigen-specific CTL and Th1 responses

a, Schematic of CFSE method to screen for polymer structures that generate strong OVA-specific CTL response. OVA was used as a model antigen (10 μg) and loaded in different polymer NPs (30 μg). b, Quantitative comparison of OVA-specific CTL responses in different NP groups (n=3 for each group) identified PC7A NP as the best candidate. OVA-specific productions of IgG1 (c) and IgG2c (d) as induced by different vaccine groups. PC7A NP produced broad CTL, Th1 and Th2 responses comparable to or better than the known adjuvants in each category. In b, c and d, representative data from three independent experiments are presented as means ± s.e.m..

Figure 2. PC7A NP improves antigen delivery and cross-presentation in APCs and stimulates CD8 T cell responses

a, Quantification of OVA-positive cells in three APC subtypes inside lymph nodes 24 h after subcutaneous injection of AF647-OVA-PC7A NP at the tail base of C57BL/6 mice (n=5). b, Schematic of detection of antigen cross-presentation in BMDCs and CD8⁺ T cell activation in vitro. c, Quantification of AF647-OVA uptake in BMDCs by flow cytometry after incubation with AF647-OVA alone, AF647-OVA-PC7A NP or AF647-OVA-PD5A NP for 4h. Mean fluorescence intensity (MFI) of AF647-OVA⁺ cells in BMDCs was determined (n=3). d, Levels of antigen presentation on H-2K^b in BMDCs induced by PC7A or PD5A NP (n=3). e, IFN-γ secretion by OT-I CD8⁺ T cells after incubating OT-I CD8⁺ T cells with BMDCs treated with different OVA-NPs (n=3). f, Representative flow dot plots of H-2k^b/SIINFEKL tetratmer staining of CD8⁺ T cells in spleen. g, Percentage of OVA (SIINFEKL) specific CD8⁺ T cells was measured by flow cytometry (n=4). In a, c–e and g, representative data from three independent experiments are presented as means ± s.e.m.. Statistical significance was calculated by Student’s t-test, ***P<0.001, **P<0.01, *P<0.05. NS, not significant.

Figure 3. PC7A NP activates APCs in draining lymph nodes and stimulates STING-dependent adaptive immune responses

a, Expression of co-stimulator CD86 on CD8α⁺ and CD8α^- DCs in inguinal lymph nodes 24 h after injection of nanovaccine (n=5 for each group). Data on macrophages and B cells are shown in Supplementary Figure 3d. b, c, Measurement of expression levels of interferon-stimulated genes (IRF7 and CXCL10) at injection site by qPCR (n=6). d, Quantitative comparison of OVA-specific CTL responses in different knockout mouse groups (n=5 for each group). IgG1 (e) and IgG2c (f) antibody titers in the serum were determined by ELISA (n=5 for each group). Data are presented as means ± s.e.m.. Statistical significance was calculated by Student’s t-test, ***P<0.001, **P<0.01, *P<0.05. NS, not significant.

Figure 4. PC7A nanovaccine inhibits tumor growth and prolongs survival in tumor bearing mice

a, Schematic of the minimalist design of PC7A nanovaccine. b, c C57BL/6 mice (n=10 per group) inoculated with 1.5×10⁵ B16-OVA tumor cells were treated with OVA peptide, PC7A nanovaccine, CpG, poly(I:C) and Alum plus peptide (0.5 μg). Tumor growth (b) and Kaplan–Meier survival curves (c) of tumor-bearing mice were shown. (d) Tumor growth inhibition study of B16F10 melanoma. C57BL/6 mice (n=10 per group) inoculated with 1.5×10⁵ B16F10 tumor cells were treated with a cocktail of tumor associated antigens (Gp100_21-41, Trp1_214-237, Trp2_173-196) in PC7A NP at specific time point indicated by the arrows. (e) Tumor growth inhibition study of MC38 colon cancer in C57BL/6 mice. Mice (n=10 per group) inoculated with 1.0×10⁶ MC38 tumor cells were treated with a cocktail of neoantigens (Reps1_P45A, Adpgk_R304M, Dpagt1_V213L) in PC7A NP, and nanovaccine was administered on day 10 and 15 in established tumors (100–200 mm³). In the HPV tumor model, tumor growth inhibition (f) and survival data (g) in C57BL/6 mice (n=10 per group) were analyzed after tumor inoculation with 1.5×10⁵ TC-1 tumor cells. In b and d–f, data are presented as means ± s.e.m.. Statistical significance was calculated by Student’s t-test, ***P<0.001, **P<0.01, *P<0.05. Statistical significance for survival analysis in c and g was calculated by the log-rank test, ***P<0.001, **P<0.01, *P<0.05.