Nanostructured lipid carriers based mRNA vaccine leads to a T cell-inflamed tumour microenvironment favourable for improving PD-1/PD-L1 blocking therapy and long-term immunity in a cold tumour model

Abstract

Background: mRNA-based cancer vaccines show promise in triggering antitumour immune responses. To combine them with existing immunotherapies, the intratumoral immune microenvironment needs to be deeply characterised. Here, we test nanostructured lipid carriers (NLCs), the so-called Lipidots®, for delivering unmodified mRNA encoding Ovalbumin (OVA) antigen to elicit specific antitumour responses.

Methods: We evaluated whether NLC OVA mRNA complexes activate dendritic cells (DCs) in vitro and identified the involved signalling pathways using specific inhibitors. We tested the anti-tumoral impact of Ova mRNA vaccine in B16-OVA and E.G7-OVA cold tumour-bearing C57Bl6 female mice as well as its synergy effect with an anti-PD-1 therapy by following the tumour growth and performing immunophenotyping of innate and adaptive immune cells. The intratumoral vaccine-related gene signature was assessed by RNA-sequencing. The immune memory response was assessed by re-challenging surviving treated mice with tumour cells.

Findings: Our vaccine activates DCs in vitro through the TLR4/8 and ROS signalling pathways and induces specific T cell activation while exhibits significant preventive and therapeutic antitumour efficacy in vivo. A profound intratumoral remodelling of the innate and adaptive immunity in association with an increase in the gene expression of chemokines (Cxcl10, Cxcl11, Cxcl9) involved in CD8⁺ T cell attraction were observed in immunised mice. The combination of vaccine and anti-PD-1 therapy improves the rates of complete responses and memory immune responses compared to monotherapies.

Interpretation: Lipidots® are effective platform for the development of vaccines against cancer based on mRNA delivery. Their combination with immune checkpoint blockers could counter tumour resistance and promote long-term antitumour immunity.

Funding: This work was supported by Inserm Transfert, la Région Auvergne Rhône Alpes, FINOVI, and the French Ministry of Higher Education, research and innovation (LipiVAC, COROL project, funding reference N° 2102992411).

Keywords: Cancer; Immune checkpoint inhibitor; Innate immunity; Memory immunity; T cells; mRNA vaccine.

Fig. 1

NLC-Ova mRNA mediates BMDC and T cell activation in vitro. (a, b) IL-6 (a) and TNFα (b) concentration in BMDC supernatant at day 1 following a 6 h or a 24 h time incubation with vehicle, NLC, NLC-Ova mRNA or Ova mRNA. (c–f) IL-6 concentration in BMDC supernatant following 3 h of pretreatment with TLR4 inhibitor TAK-242 at three different concentrations (1.25 μM, 2.5 μM, and 5 μM) (c) or TLR8 inhibitor CU-CPT9a (2.5 μM, 5 μM, and 10 μM) (d) or following 1 h of pretreatment with ROS-dependent NOX inhibitor DPI (0.1 μM, 1 μM, and 10 μM) (e) or mitochondrial ROS (mROS) inhibitor S3QEL2 (1 μM, 10 μM, and 30 μM) (f) and 24 h of NLC-Ova mRNA incubation. Mean ± SEM of 4–7 independent experiments where each dot represents the mean of 2 replicates. (g–j) CD8⁺ T cells (g, h) or CD4⁺ T cells (i, j) from OT-I or OT-II spleen respectively were cocultured for 48 h with BMDCs treated with vehicle, NLC, NLC-cherry mRNA or NLC-Ova mRNA. Frequencies of CD69⁺ (g, i) and IFNγ⁺ (h, j) were assessed by flow cytometry. Mean ± SEM of four independent experiments where each dot represents the mean of 2–3 replicates. P values (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001) determined by Kruskal–Wallis test with Dunn's post hoc testing (a, d, g–j) or one-way ANOVA with Dunnett's post hoc testing (b, c, e, f).

Fig. 2

Antitumour effect of NLC-Ova mRNA complex as a preventive vaccine. (a–c) Tumour growth in NLC-Ova mRNA immunised mice injected i.d. with B16-OVA melanoma cells. NLC-Cherry mRNA was used for the control group (b). Representative pictures of the B16-OVA tumour from NLC-Ova mRNA or NLC-Cherry mRNA treated mice at sacrifice (c). (d–i) CD8⁺ T cell analysis from spleen of B16-OVA tumour-bearing mice immunised with NLC-Ova mRNA or NLC-Cherry mRNA. Following ex vivo restimulation with OVA peptides, IL-2 (d), IFNγ (e), GzmB (f), TNFα (g) production, IFNγ and TNFα coproduction (h), and PD-1 expression (i) were assessed by flow cytometry. Mean ± SEM of N = 6 mice per group. P values (∗∗p < 0.01) determined by Mann–Whitney test (b–i).

Fig. 3

Antitumour effect of NLC-Ova mRNA complex as a therapeutic vaccine. (a, b) Tumour growth in mice injected with B16-OVA melanoma cells (a) or E.G7-OVA lymphoma cells (b) and then treated with NLC-Ova mRNA or NLC-irrelevant mRNA. (c–j) Intratumoral frequency of CD45⁺ cells in live cells (c), CD8⁺ T cells in CD45⁺ cells (d), number of CD8⁺ T cells per mg of B16-OVA tumour tissue (e) from NLC-Ova mRNA or NLC-Cherry mRNA-treated mice. Surface of CD8⁺ T cells (f) and PD-1⁺ T cells (g) analysed by IHC for each tumour group. Intratumoral frequency of CD8⁺ T cells expressing CD69 (h) and coexpressing IFNγ and TNFα (i) or IFNγ and GzmB (j) from B16-OVA tumour-bearing mice treated with NLC-Ova mRNA or NLC-irrelevant mRNA following OVA peptide stimulation. (k) Correlations between intratumoral CD69⁺CD8⁺ T cells frequency or CD69⁺CD4⁺ T cells frequency and tumour weight at day 17 in B16-OVA-bearing tumour mice treated with NLC-Ova mRNA or NLC-Cherry mRNA. (l) Survival in B16-OVA tumour-bearing mice treated i.p. with NLC-Egfp mRNA or NLC-Ova mRNA as well as with anti-CD8β or its control IgG1. Mean ± SEM of N = 6 mice per group. P values (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001) determined by Mann–Whitney test (a–j) or Spearman r correlation test (k) or log-rank (Mantel–Cox) test (l).

Fig. 4

NLC-Ova mRNA vaccine produces an inflamed immune gene signature. Mice bearing B16-OVA tumours were treated with NLC-Egfp mRNA or LNP-Ova mRNA twice and analysed by RNA-seq. (a) Volcano plot showing the fold change (log2, x-axis) and statistical significance (−log10 Q value, y-axis) of differentially expressed genes (DEGs) (log2 fold change >2 and Q value < 0.005). (b) Top 10 of selective enrichment of biological pathways following GSEA based on 487 DEGs between NLC-Egfp mRNA and NLC-Ova mRNA groups (log2 fold change >2 and Q value < 0.005). (c, d) Enrichment plot of indicated signatures. Normalised Enrichment score (NES), False Discovery Rate (FDR) Q value shown for the two gene sets. (e) KEGG pathway classification based on 487 DEGs between NLC-Egfp mRNA and NLC-Ova mRNA groups (log2 fold change >2 and Q value < 0.005). (f, g) Heatmaps of genes related to “T cell receptor signaling pathway” (f) and “chemokine signaling pathway” (g) following the selection of the most significative pathways from a KEGG pathway relationship network based on the 123 genes related to the “immune system” pathway. N = 4 mice per group.

Fig. 5

NLC-Ova mRNA vaccine cooperates with anti-PD-1 to suppress B16-OVA tumour growth. (a, b) Tumour growth (a) and survival (b) in mice injected with B16-OVA tumour cells and treated i.p. with NLC-Ova mRNA or NLC-Egfp mRNA as well as with anti-PD-1 or its control IgG2a. Mean ± SEM of N = 12 mice per group. P values (∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001) determined by Kruskal–Wallis test with Dunn's post hoc testing (a, D17) and Mann–Whitney test (a, D23) or log-rank (Mantel–Cox) test (b). (c–m) Intratumoral frequency of CD86⁺ DCs (CD11c⁺MHCII⁺) (c), CD86⁺ macrophages (CD11b⁺F4/80⁺) (d), CD206⁺ macrophages (e) and proinflammatory monocytes (CD11b⁺Ly6C^high) (f) PD-L1 expression (MFI) in PD-L1⁺ DCs (g), PD-L1⁺ macrophages (h) and PD-L1⁺ inflammatory monocytes (CD11b⁺Ly6C^high) (i); intratumoral frequency of CD8⁺ T cells coexpressing IFNγ and GzmB (j), expressing PD-1 (k), coexpressing PD-1 and TIM-3 (l) and intratumoral frequency of CD4⁺ T cells expressing GzmB (m) from B16-OVA tumour-bearing mice treated with NLC-Ova mRNA or NLC-Egfp mRNA as well as with anti-PD-1 or IgG2a. Mean ± SEM of N = 6 mice per group. P values (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001) determined by two-way ANOVA (mRNA vaccine effect (mRNA) anti-PD-1 treatment effect (aPD-1) and their interaction (X)) with Dunnett's post hoc testing (c–g, i–m) or Kruskal–Wallis test with Dunn's post hoc testing (h). NS, not significant.

Fig. 6

Protection from tumour re-challenge in survivor mice treated with NLC-Ova mRNA ± anti-PD-1. (a) Tumour growth in NLC-Ova mRNA- (orange dot surrounded by a black circle) or NLC-Ova mRNA + anti-PD-1- (orange dot) treated surviving mice challenged with a second injection of B16-OVA cancer cells 103 days following the first B16-OVA tumour cell injection. Control mice (black dot) were similar age than challenged mice and were injected with the same number of B16-OVA tumour cells (2 × 10⁵). (b–i) Frequency of CD8⁺ T cells expressing IFNγ (b, c), TNFα (d, e), coexpressing IFNγ and TNFα (f); frequency of OVA-specific CD8⁺ T cell (g); expression level of CD86 (MFI) in CD86⁺ DCs (h) and frequency of B cells expressing CD86 and MHC-II (i) into the spleens from control and challenged B16-OVA tumour-bearing mice following OVA peptide stimulation. (j, k) Tumour growth (j) and weight (k) in NLC-Ova mRNA- or NLC-Ova mRNA + anti-PD-1-treated surviving mice challenged with an injection of B16 cancer cells 113 days following the first B16-OVA tumour cell injection. (l, m) Intratumoral frequency of NK cells expressing CD69 from control and challenged B16 tumour-bearing mice (l) as well as correlation between B16 intratumoral frequency of CD69⁺ NK cells and tumour weight at day 14 (m). Mean ± SEM of N = 9 mice per group (a–g) or N = 5–6 mice per group (h–m). P values (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001) determined by Mann–Whitney test (a–l) or Spearman r correlation test (m). NS, not significant.