Intratumoral delivery of lipid nanoparticle-formulated mRNA encoding IL-21, IL-7, and 4-1BBL induces systemic anti-tumor immunity

Intratumoral injection of LNP-formulated mRNA leads to efficient protein expression in myeloid and cancer cells

Lipid-based nanoparticles are highly effective carriers to enhance mRNA delivery in vivo. The ionizable lipid is considered the most critical component within the LNP, as it mediates mRNA encapsulation and the efficiency of endosomal escape. Recently, we have reported the design of the ionizable cationic lipid S-Ac7-DOG that combines improved expression levels with reduced reactogenicity upon intramuscular injection (Supplementary Fig. 1A)^33,34. To evaluate whether LNP-mediated mRNA delivery using the same LNP composition leads to the expression of the target protein in vitro, we developed and characterized LNPs carrying mRNA encoding the cell surface glycoprotein thymus cell antigen 1a (Thy1.1) (Supplementary Fig. 1B). We then transfected the MC38 colon carcinoma cell line with the LNP-formulated Thy1.1 mRNA and quantified Thy1.1 expression after one day using flow cytometry. Thy1.1 LNP transfected MC38 cells and induced Thy1.1 expression in almost 100% of MC38 cells (Supplementary Fig. 1C). To assess the in vivo biodistribution of the target protein and whether LNP-mediated mRNA delivery enhanced its expression relative to naked mRNA, we injected subcutaneous MC38 colon carcinoma tumors with either mRNA encoding the firefly luciferase (FLuc mRNA) or LNPs carrying the firefly luciferase mRNA (FLuc LNP). Intratumoral delivery of LNP-formulated FLuc mRNA induced higher luciferase activity than that of naked FLuc mRNA as analyzed by in vivo bioluminescent imaging (Fig. 1A). However, as previously reported³⁵, accumulation of FLuc LNP was also observed in the liver, albeit to a lower extent than in the tumor (Fig. 1A). Next, we sought to identify the cells expressing the target protein within the TME. To this end, we injected either naked or LNP-formulated Thy1.1 mRNA intratumorally into subcutaneous MC38 tumors and quantified Thy1.1 expression after one day using flow cytometry (Fig. 1B). LNP mediated Thy1.1 mRNA delivery resulted in increased expression compared to naked Thy1.1 mRNA in both CD45⁺ (immune) and CD45^– (non-immune) cells (Fig. 1C). Within the CD45⁺ compartment, Thy1.1 expression was restricted to CD11b⁺ myeloid cells and in particular to tumor-associated macrophages (TAMs), monocytes, and conventional dendritic cells type 2 (cDC2s) (Fig. 1D and Supplementary Fig. 1D). Here, we defined three different subsets of TAMs, based on their expression of major histocompatibility complex (MHC)-II and macrophage mannose receptor (MMR), the latter marker being associated with an M2-like phenotype, and found that MMR⁺ TAMs were enriched within the Thy1.1⁺ TAMs (Supplementary Fig. 1E). Accordingly, Thy1.1 expression was higher in M2-polarized bone marrow-derived macrophages (BMDM) compared to M1-polarized BMDMs incubated with Thy1.1 mRNA LNPs (Supplementary Fig. 1F). Since TAMs and monocytes are the most abundant myeloid cells within MC38 tumors (Supplementary Fig. 1G), we speculated that their high frequency within the Thy1.1⁺ compartment could be merely due to their high abundance in the TME rather than their superior capacity to take up the LNPs. To take this into account, we quantified the frequency of Thy1.1⁺ cells within TAMs, monocytes, and cDC2s and found that cDC2s showed the highest intrinsic uptake capacity of the Thy1.1 LNP and consequent Thy1.1 protein expression (Fig. 1E). To assess whether LNP uptake impacts cDC2 activation, we determined the expression of several cDC activation markers including CCR7, CD80, CD40, CD83, programmed cell death ligand (PD-L) 1, and MHC-II in Thy1.1⁺ and Thy1.1^– cDC2s (Fig. 1F, G and Supplementary Fig. 1H–K). CCR7 and CD80 levels increased significantly in Thy1.1⁺ cDC2s, suggesting that LNP uptake and mRNA translation might induce cDC2 activation and migration toward the tumor-draining lymph node (tdLN). In this respect, we observed that in the tdLN, cDC2s were the most abundant Thy1.1⁺ cells and that approximately 90% of Thy1.1⁺ cDC2s in the tdLN had a migratory phenotype (CD11c^int MHC-II^hi CCR7⁺), suggesting that they might have migrated from the tumor to the tdLN upon taking up the Thy1.1 LNP (Fig. 1H and Supplementary Fig. 1L). Importantly, the frequency of Thy1.1⁺ cells in the tdLN and spleen was drastically lower compared to that of the tumor (Fig. 1I), indicating limited leakage of Thy1.1 LNP into the secondary lymphoid tissues. Of note, we identified a minor population of Thy1.1⁺ CD19⁺ B cells in the tdLN and spleen, which was similarly observed in vehicle-treated mice; therefore, we excluded this population from our analysis (Supplementary Fig. 1M). Next, we injected Thy1.1 LNP intratumorally into subcutaneous MC38 tumors to assess the relative Thy1.1 expression in the liver one day after the injection (Fig. 1B). The frequency of Thy1.1⁺ cells in the liver was significantly lower than in the tumor (Supplementary Fig. 2A). In the liver, Thy1.1⁺ cells were mostly CD45⁺ myeloid cells, particularly monocytes, neutrophils and to a lower extent Kupffer cells (Supplementary Fig. 2B–E).

A Images and bar graph show in vivo bioluminescence of luciferin over 72 h in MC38 tumor following a single intratumoral administration of vehicle (n = 4 animals), naked luciferase mRNA (n = 5 animals) or LNP-formulated luciferase mRNA (n = 5 animals). B Schematic outline of experimental procedures. C Representative flow cytometry plots and bar graphs show the frequency of Thy1.1⁺ CD45⁺ and Thy1.1⁺ CD45^– cells in subcutaneous MC38 tumors 24 h post intratumoral injection of vehicle (n = 8 animals), naked Thy1.1 mRNA (n = 5 animals), or Thy1.1 LNP (n = 10 animals). D Gating strategy and bar graph show the frequency of myeloid cell subsets within Thy1.1⁺ CD45⁺ cells in the tumor (n = 10 animals). E Representative flow cytometry plots and graph show the frequency of Thy1.1⁺ cells within TAMs, monocytes, and cDC2s in the tumor (n = 10 animals). Graphs show gMFI of (F) CCR7 and (G) CD80 in Thy1.1⁺ and Thy1.1^– cDC2s in the tumor (n = 5 animals). H Representative flow cytometry plots and scatter plot show the frequency of migDC and resDC within Thy1.1⁺ cDC2s in the tdLN (n = 5 animals). I Bar graph shows the frequency of live Thy1.1⁺ cells in the tumor, tdLN, and spleen (n = 10 animals). Bars and horizontal lines show mean. Error bars indicate SEM. A Two-way ANOVA followed by Šídák’s multiple comparisons test, C, I one-way ANOVA followed by post hoc Tukey’s multiple comparisons test, E repeated measures one-way ANOVA followed by Holm-Šídák’s multiple comparisons test, F, G one-tailed Wilcoxon matched pairs signed rank test and (H) one-tailed unpaired t test were performed. Data shown in (B–D), and (H) are pooled from two independent experiments. Source data are provided as a Source Data file. gMFI, geometric mean fluorescence intensity; SEM, standard error of mean; TAMs, tumor-associated macrophages; tdLN, tumor-draining lymph nodes.

Full size image

Triplet LNP eradicates tumors across multiple preclinical models

Having demonstrated that mRNA delivery via LNPs elicited a higher protein expression than naked mRNA in tumors, we sought to assess the therapeutic potential of LNPs carrying mRNA encoding immunostimulatory proteins. Given the reported anti-tumor efficacy of IL-21^{11,12,13,14,15,16}, we first developed LNPs carrying IL-21 mRNA. Following a single intratumoral injection of IL-21 LNP, we observed a profound increase in the protein levels of IL-21 in the tumor and serum presumably due to its leakage from the tumor into the circulation (Fig. 2A, B and Supplementary Fig. 3A). Three intratumoral injections of IL-21 LNP showed a moderate efficacy against MC38 tumors, inducing complete tumor regression in 4 out of 8 mice (Supplementary Fig. 3B, C). To enhance the therapeutic efficacy of IL-21, we sought to combine it with other potentially synergistic immunostimulatory molecules. IL-7 has been shown to synergize with IL-21 in vitro and in vivo, promoting CD8⁺ T-cell proliferation and cytotoxicity^36,37,38. 4-1BB has emerged as an attractive immunotherapy target amongst other molecules, such as OX40 and CD27, due to its role in enhancing CD8⁺ T-cell survival and cytokine production²⁸. To this end, we developed LNPs carrying either IL-7 or 4-1BBL mRNA and assessed their protein levels in the tumor and serum post a single intratumoral injection (Fig. 2A, C, D and Supplementary Fig. 3D, E). Next, we evaluated the therapeutic efficacy of IL-21, IL-7 and 4-1BBL LNPs alone and their combinations to assess their potential synergy (Fig. 2E, F). While IL-21 LNP as a monotherapy was able to abrogate MC38 tumor growth in 3 out of 7 mice, IL-7 and 4-1BBL alone had no effect on tumor growth. IL-7 synergized with IL-21 and resulted in complete tumor regression in 5 out of 7 mice (Fig. 2F). In contrast, in IL-21/4-1BBL LNP-treated mice, only 1 out of 7 mice showed complete tumor regression, suggesting a potentially lower anti-tumor activity compared to IL-21 alone (Fig. 2F). Interestingly, mice treated with IL-7/4-1BBL LNP showed delayed tumor growth in comparison to IL-7 and 4-1BBL monotherapies and completely abrogated tumor growth in 1 out of 7 mice (Fig. 2F). More importantly, the combination of IL-21, IL-7 and 4-1BBL (i.e. Triplet LNP) showed the highest therapeutic efficacy and led to 6 out of 7 complete regressors (Fig. 2F).

A Schematic outline of experimental procedures. Scatter plots show the concentration of (B) IL-21 and (C) IL-7 in MC38 tumor supernatants 4 h post intratumoral injection of vehicle (n = 8 animals), control LNP (n = 8 animals), or cytokine LNP (n = 4 animals). D Representative flow cytometry plots and bar graph show the frequency of live 4-1BBL⁺ cells in subcutaneous MC38 tumor 24 h post intratumoral injection of vehicle (n = 4 animals), control LNP (n = 4 animals), or 4-1BBL LNP (n = 5 animals). E Schematic outline of experimental procedures. F MC38 tumor individual growth curves of mice treated with vehicle, control LNP, IL-21 LNP, IL-7 LNP, 4-1BBL LNP, IL-21/IL-7 LNP, IL-21/4-1BBL LNP, IL-7/4-1BBL LNP and Triplet LNP (n = 7 animals/group). Vertical dotted lines indicate intratumoral injections. G Schematic outline of experimental procedures. H E0771 tumor individual growth curves of mice treated with vehicle (n = 12 animals), control LNP (n = 11 animals), Triplet LNP (n = 12 animals), anti-PD1 (n = 12 animals) and Triplet LNP/anti-PD1 (n = 12 animals). Vertical dotted lines indicate intratumoral injections. I Schematic outline of experimental procedures. J Survival curve of subcutaneous B16F10 tumor-bearing mice treated with vehicle, isotype control, control LNP/isotype control, Triplet LNP/isotype control, anti-PD1, control LNP/anti-PD1, and Triplet LNP/anti-PD1 (n = 10 animals/group). B–D One-way ANOVA followed by post hoc Tukey’s multiple comparisons test and (J) log-rank (Mantel-Cox) test were performed. Data shown in (H) is pooled from two independent experiments. Bars and horizontal lines show mean. Error bars indicate SEM. Source data are provided as a Source Data file. CR, complete regressor; SEM, standard error of mean.

Full size image

Having demonstrated the therapeutic efficacy of the Triplet LNP in the MC38 tumor model, we sought to assess its efficacy in ICB-resistant models. Triple-negative breast cancer (TNBC) is an aggressive disease with a poor prognosis due to its limited response to the available therapeutic options, including ICB³⁹. Therefore, we evaluated the therapeutic efficacy of IL-21 LNP, IL-7 LNP, 4-1BBL LNP, IL-21/IL-7 LNP and Triplet LNP in mice bearing orthotopic E0771 TNBC tumors (Supplementary Fig. 3F, G). In contrast to the MC38 tumor model, the E0771 model was susceptible to 4-1BBL monotherapy as complete tumor regression was observed in 3 out 7 mice (Supplementary Fig. 3G). The combination of IL-7 and IL-21 promoted complete tumor regression in 4 out 7 E0771 tumor-bearing mice, while the Triplet LNP led to 6 out 7 complete regressors, suggesting that 4-1BBL is essential for the therapeutic efficacy of the Triplet LNP, particularly against E0771 tumors (Supplementary Fig. 3G). Then, we compared the therapeutic efficacy of the Triplet LNP to the ICB anti-PD1 in E0771 tumor-bearing mice (Fig. 2G, H). The Triplet LNP alone was sufficient to induce complete tumor regression in a notable 10 out of 12 E0771 tumor-bearing mice, strongly outperforming anti-PD1 (1 of 12 complete regressors) (Fig. 2H). Remarkably, combining the Triplet LNP treatment with anti-PD1 completely eradicated the E0771 tumors (12 out of 12 complete regressors) (Fig. 2H). Moreover, we investigated the therapeutic efficacy of the Triplet LNP and anti-PD1 in the subcutaneous B16F10 melanoma model. While both the Triplet LNP and anti-PD1 monotherapy failed to improve survival, the Triplet LNP/anti-PD1 combination significantly enhanced survival with 4 out of 10 complete regressors (Fig. 2I, J), showcasing the synergistic potential of the Triplet LNP and anti-PD1 in non-responsive models.

Triplet LNP displays a favorable safety profile

Considering the established dose-limiting hepatotoxicity of 4-1BB agonists^29,30,31,32, we sought to assess the potential hepatotoxicity of the Triplet LNP treatment. Generally, all treated mice tolerated the therapy well, experienced no weight loss, and showed no signs of acute toxicity. Serum alanine transaminase (ALT) and aspartate transaminase (AST) activities measured 4 and 24 h after the first injection and as well as four days following the third injection were not elevated in Triplet LNP-treated mice (Supplementary Fig. 4A–E). Flow cytometry analysis of the livers four days following the third dose revealed a slight increase in the frequency of CD45⁺ cells in the Triplet LNP-treated mice, but no significant impact on the frequencies of monocytes or T cells, including CD4⁺ T cells and CD8⁺ T cells (Supplementary Fig. 4F–K). Additionally, we performed hematoxylin and eosin (H&E) and Sirius Red stainings to assess the effects of the Triplet LNPs on the microanatomy of the liver four days and eight weeks (complete regressors) following the third injection (Supplementary Fig. 5). Compared to vehicle-treated mice, a slight increase in lobular inflammation was seen in LNP-treated mice four days after the third dose, which tended to be more elevated in the Triplet LNP-treated group (Supplementary Fig. 5A and Supplementary Table 1). No increase in portal or biliary inflammation was observed at this timepoint (Supplementary Fig. 5A and Supplementary Table 1). The Triplet LNP treatment also resulted in a slightly elevated fibrosis score. Nonetheless, eight weeks after the third dose, Triplet LNP-treated complete regressors did not show signs of increased liver inflammation or fibrosis in comparison to vehicle-treated mice (Supplementary Fig. 5A, B and Supplementary Table 1). Overall, these data indicate that intratumoral administration of the Triplet LNP is well tolerated and does not elicit profound or long-lasting hepatoxicity, in sharp contrast to what has been reported for systemic treatment with 4-1BB agonistic antibodies⁴⁰.

Triplet LNP eradicates tumors by promoting CD8⁺ T-cell infiltration and activation

We investigated the impact of the Triplet LNP on the immune effector cells within the tumor two days following the second intratumoral injection (Fig. 3A, B and Supplementary Fig. 6A). The Triplet LNP tremendously increased the frequency of CD8⁺ T cells and consequently, the frequency of TAMs decreased, though the relative frequency of CD11c^hi MHC-II⁺ TAMs remained constant across the different treatments (Fig. 3C, D and Supplementary Fig. 6B). Despite the increase in the frequency of CD8⁺ T cells, the Triplet LNP did not improve their proliferative capacity, however, it increased the proportion of CD44^hi CD62L^lo effector CD8⁺ T cells (Fig. 3E and Supplementary Fig. 6C). Moreover, the Triplet LNP significantly induced cytotoxic granzyme B⁺ CD8⁺ T cells and enhanced the capacity of CD8⁺ T cells to produce IFN-γ and TNF-α (Fig. 3F, G). Although the Triplet LNP had no impact on the abundance of NK cells in the tumor, the frequency of granzyme B⁺ and IFN-γ⁺ TNF-α⁺ NK cells increased significantly in the tumor (Fig. 3H–J). To dissect the role of the separate components of the Triplet LNP, the changes within the TME were assessed two days following the second intratumoral injection of either IL-21 LNP, IL-21/IL-7 LNP, IL-21/4-1BBL LNP, or Triplet LNP (Fig. 3A). While IL-21 was sufficient to increase the frequency of granzyme B⁺ CD8⁺ T cells in the tumor, only the combination of IL-21 with IL-7 improved CD8⁺ T-cell infiltration into the tumor two days following the second intratumoral injection (Supplementary Fig. 6D, E). Interestingly, the presence of 4-1BBL was necessary to maintain the levels of CD11c^hi MHC-II⁺ pro-inflammatory TAMs in the tumor (Supplementary Fig. 6F).

A Schematic outline of experimental procedures. B Pie charts show the mean frequency of immune subsets within CD45⁺ cells in the tumor of vehicle-, control LNP- or Triplet LNP-treated mice quantified using flow cytometry (n = 7 animals/group). Bar graphs show the frequency of (C) CD8⁺ T cells, (D) TAMs, (E) CD44^hi CD62L^lo CD8⁺ T cells, (F) Granzyme B⁺ CD8⁺ T cells, (G) IFN-γ⁺ TNF-α⁺ CD8⁺ T cells, (H) NK cells (I) Granzyme B⁺ NK cells within MC38 tumors treated with vehicle, control LNP or Triplet LNP (n = 7 animals/group) and (J) IFN-γ⁺ TNF-α⁺ NK cells within MC38 tumors treated with vehicle (n = 7 animals), control LNP (n = 7 animals) or Triplet LNP (n = 6 animals). K Representative flow cytometry plots and graph show the frequency of CD39⁺ CX3CR1⁺ CD8⁺ T cells over time in the blood of vehicle (n = 5 animals), control LNP (n = 5 animals), and Triplet LNP-treated mice (n = 6 animals). MC38 tumor growth curves upon (L) isotype or αNK1.1 administration (isotype/vehicle n = 5 animals; isotype/control LNP n = 5 animals; isotype/triplet LNP n = 6 animals; αNK1.1/vehicle n = 5 animals; αNK1.1/control LNP n = 5 animals; αNK1.1/triplet LNP n = 6 animals) and (M) isotype or αCD8 administration (isotype/vehicle n = 9 animals; isotype/control LNP n = 9 animals; isotype/triplet LNP n = 12 animals; αCD8/vehicle n = 10 animals; αCD8/control LNP n = 10 animals; αCD8/triplet LNP n = 12 animals). C–J One-way ANOVA followed by post hoc Tukey’s multiple comparisons test and (K–M) repeated measures two-way ANOVA followed by post hoc Tukey’s multiple comparisons test were performed. Data shown in (B–J) are representative of three independent experiments. Data shown in (K) is representative of two independent experiments. Data shown in (M) is pooled from two independent experiments. Bars and horizontal lines show mean. Error bars indicate SEM. Source data are provided as a Source Data file. SEM, standard error of mean; TAMs, tumor-associated macrophages.

Full size image

Next, we evaluated the impact of the Triplet LNP on the immune landscape of the E0771 tumors two days following the second intratumoral injection (Supplementary Fig. 6G). The Triplet LNP treatment enhanced CD8⁺ T-cell infiltration in the tumor, which was accompanied by a shift toward an activated effector memory state and a higher production of granzyme B, IFN-γ, and TNF-α (Supplementary Fig. 6H–L). Moreover, the Triplet LNP reduced the frequency of TAMs, with no significant changes in the frequency of MHC-II⁺ TAMs (Supplementary Fig. 6M, N).

The observed increase in the frequency of CD8⁺ T cells in the Triplet LNP-treated MC38 tumor-bearing mice was not restricted to the TME. In the blood of the Triplet LNP-treated mice, a consistent increase in the frequency of T cells, in particular CD8⁺ T cells, was observed as of five days post the first intratumoral injection (Supplementary Fig. 7A). It has been shown that the frequency of the CX3CR1⁺ subset of circulating CD8⁺ T cells correlates with response to immune checkpoint blockers and survival of MC38 tumor-bearing mice and non-small cell lung carcinoma (NSCLC) patients, hence it can be used as a blood-based biomarker of response to immunotherapy^41,42. Interestingly, the frequency of tumor-matching CD8⁺ T cells within the CD8⁺ T-cell population in the blood, identified here as CD39⁺ CX3CR1⁺ CD8⁺ T cells, increased significantly in the Triplet LNP-treated mice in comparison to the control cohorts (Fig. 3K). Hence, the presence of CD39⁺ CX3CR1⁺ CD8⁺ T cells in the blood could potentially be used as a prognostic marker for the response to Triplet LNP therapy. Accordingly, the frequency of CD4⁺ T cells in the Triplet LNP-treated mice slightly decreased over time compared to the control cohorts (Supplementary Fig. 7B). Next, we sought to determine whether NK cells or CD8⁺ T cells are required for the therapeutic efficacy of the Triplet LNP. Using anti-NK1.1 and anti-CD8-depleting antibodies, we found that while the partial depletion of NK cells did not affect the therapeutic efficacy of the Triplet LNP, the depletion of CD8⁺ T cells abolished the therapeutic effect completely, indicating that the therapeutic efficacy of the Triplet LNP is CD8⁺ T cell-dependent (Fig. 3L, M and Supplementary Fig. 7C, D).

Triplet LNP alters the relative abundance of CD8⁺ T-cell subsets within the TME

To decipher the impact of the Triplet LNP on the different CD8⁺ T-cell subsets in the TME, we performed CITE-seq on the immune-cell compartment of the tumor. In line with our flow cytometry results, the T-cell compartment increased profoundly in tumors of the Triplet LNP-treated mice compared to vehicle and control LNP-treated mice (Fig. 4A and Supplementary Fig. 8A, B). High-resolution subclustering of the T-cell compartment based on the transcriptome and selected surface proteins revealed twelve distinct clusters of which the proportions of CD8T Prf1 Lag3 hi, CD8T Ccl3 Nr4a3 and CD8T Ccr2 Gzma clusters increased in the tumors of the Triplet LNP-treated mice (Fig. 4B and Supplementary Fig. 8C, D). In the CD8T Prf1 Lag3 hi cluster, which showed an 8-fold increase upon treatment with the Triplet LNP, genes associated with T-cell exhaustion (Entpd1, Lag3, Pdcd1, Havcr2) and effector function (Prf1, Gzmf, Gzmc, Ccl3, Ccl4) were upregulated (Fig. 4C and Supplementary Fig. 8E). Accordingly, the expression of the respective T-cell exhaustion proteins (CD39, CD223 (LAG3), PD1, CD366 (TIM-3)) strongly correlated with that of the genes (Fig. 4D and Supplementary Fig. 9A). Gene ontology (GO) enrichment analysis of the upregulated genes in this CD8T Prf1 Lag3 hi cluster showed enrichment in GO terms associated with the regulation of T-cell activation and cytotoxicity (Fig. 4E). In the CD8T Ccl3 Nr4a3 cluster, genes of several proinflammatory cytokines and chemokines (Tnf, Ifng, Ccl3, Ccl4, Xcl1) were upregulated, suggesting their potential role in attracting and activating other immune cells including T cells, NK cells and DCs (Fig. 4C). The CD8T Ccr2 Gzma cluster exhibited a limited set of differentially upregulated genes, which included Ccr2, Gzma, and Ccl5 (Fig. 4C). To identify the phenotypic state of these clusters, we projected our T-cell cluster onto the tumor-infiltrating T-cell atlas by Andreatta et al.⁴³, which identified CD8T Prf1 Lag3 hi, CD8T Ccl3 Nr4a3 and CD8T Ccr2 Gzma as exhausted, progenitor exhausted and effector memory, respectively (Supplementary Fig. 9B). Despite having a tremendous impact on the abundance of different CD8⁺ T-cell states in the tumor, the Triplet LNP treatment had a minimal impact on the overall cytotoxic and exhaustion signatures within these subsets (Fig. 4F).

A UMAP plot of CITE-seq dataset containing 30208 CD45⁺ sorted cells from MC38 tumors of vehicle, control LNP, and Triplet LNP-treated mice (n = 5 animals/group). B High-resolution subclustering of the T-cell compartment of the dataset is shown. Bars show the frequency of different T-cell subsets in the tumor dataset across different treatments. C Heatmap of the top 10 differentially expressed genes in the CD8T Prf1 Lag3 hi, CD8T Ccl3 Nr4a3 and CD8T Ccr2 Gzma clusters. D Heatmap of the top 10 differentially expressed proteins in the CD8T Prf1 Lag3 hi, CD8T Ccl3 Nr4a3 and CD8T Ccr2 Gzma clusters. E GO term enrichment analysis of the differentially upregulated genes in the CD8T Prf1 Lag3 hi cluster in comparison to all other T-cell clusters in the tumor dataset. F Violin plots show combined mean expression values for the indicated genes (score) for the cytotoxic T cell markers (Gzma, Gzmb, Prf1, Tnf, Ifng, Cxcr6) or exhaustion markers (Pdcd1, Ctla4, Tigit, Lag3, Havcr1, Tox, Nrp1) in cells of the CD8T Prf1 Lag3 hi, CD8T Ccl3 Nr4a3 and CD8T Ccr2 Gzma clusters in vehicle-, control LNP- and Triplet LNP-treated mice. One-way ANOVA followed by post hoc Tukey’s multiple comparisons test was performed. Source data for (F) are provided as a Source Data file. GO, gene ontology; UMAP, uniform manifold approximation and projection.

Full size image

Triplet LNP improves myeloid-CD8⁺ T-cell interactions

To identify the cellular targets of the Triplet LNP treatment, we assessed the expression levels of Il21r, Il7r, and Tnfrsf9 (encoding 4-1BB receptor) across the different immune cell subsets in the tumor. Il21r transcripts were expressed at varying levels in B cells, T cells, DCs, TAMs, and monocytes, while the expression of Il7r and Tnfrsf9 was restricted mostly to DCs and lymphocytes (Supplementary Fig. 9A). Therefore, we speculated that the Triplet LNP treatment might have direct and indirect effects on the abundance and activation of different myeloid cell subsets. We subclustered the DC compartment into five clusters in which the migDC cluster was slightly more abundant in the Triplet LNP-treated mice (Fig. 5A and Supplementary Fig. 10B, C). Additionally, the Triplet LNP increased the expression of genes associated with DC maturation in the cDC1 and cDC2 clusters without altering genes associated with DC regulation/exhaustion (Fig. 5B–D). It has been recently shown that successful immunotherapy led to the transient increase and expansion of a distinct neutrophil subset with an IFN-stimulated gene signature⁴⁴. In line with these findings, we observed an expansion in two neutrophil clusters with IFN-stimulated gene signatures upon the Triplet LNP treatment, namely Cxcl2hi Gbp4hi and Cxcl2hi IFN neutrophils (Fig. 5E). The TAM/Monocyte compartment was clustered into six subclusters based on the transcriptome and surface protein markers in which the relative abundance of the Mac immature cluster increased upon the Triplet LNP treatment (Supplementary Fig. 10D–F). Accordingly, the proportion of the Mac hypoxic and Mac prolif decreased (Supplementary Fig. 10D). Here, the Triplet LNP treatment elevated the transcripts of genes associated with an M1-like phenotype in the Mac immature, Mac MHCII-hi, Mac hypoxic and Mac prolif clusters (Supplementary Fig. 10G, H). Altogether, our data indicate that the Triplet LNP treatment led to notable changes in the myeloid compartment in terms of both the abundance of distinct subsets and their gene expression profile. Next, to determine the effect of the treatment on the myeloid-CD8⁺ T-cell interactions, we performed a differential Nichenet analysis to predict the niche-specific ligand-receptor pairs between the different myeloid cell subsets and CD8⁺ T cells in the control niche (vehicle + control LNP) and Triplet LNP niche. The differential Nichenet approach predicts ligand-receptor pairs that are both differentially expressed and show enrichment of the expression of their known target genes between conditions. From the top 50 ligand-receptor interactions in the Triplet LNP niche, the most numerous links were found with ligands specific for the Mac Folr2, migDC and Neutro IFN clusters (Fig. 5F). Among the top differentially expressed ligands in the Triplet LNP niche were the migDC-specific co-stimulatory molecule Cd86 and the proinflammatory cytokines Il18 (enriched in Mono and Mac Folr2) and Cxcl9 (upregulated in Mac MHCII-hi) (Fig. 5F). The predicted target genes of the top Triplet LNP-specific ligand-receptor pairs included Ccl4, Ccl3, Ifng, Prf1, Cxcl9, Nr4a1, Nr4a2, and Il2ra, which are associated with CD8⁺ T-cell effector functions and activation (Fig. 5F). These results suggest that upon Triplet LNP therapy, distinct myeloid populations contribute to the activation of cytotoxic CD8⁺ T cells in the TME. Nevertheless, the depletion of neutrophils or TAMs using anti-Ly6G/anti-rat antibodies or the CSF1R inhibitor PLX5622, respectively, had no impact on the therapeutic efficacy of the Triplet LNP (Fig. 5G, H and Supplementary Fig. 11A, B).

A UMAP plot shows high-resolution clustering of the DC compartment in the CITE-seq dataset of the tumor. Bars show the frequency of different DC subsets in the tumor dataset across different treatments. B Dot plot highlights differentially expressed genes associated with maturation, regulation, and migration in distinct DC clusters of the tumor CITE-seq dataset across different treatments. Dot size represents the percentage of cells expressing the gene and color gradient represents average scaled expression within a cell cluster. Violin plots show combined mean expression values for the indicated genes (score) for (C) maturation markers (Cd40, Cd80, Cd86, Relb, Cd83) and (D) regulation (Axl, Ccl19, Ccl22, Aldh1a1) in cells of the DC clusters in vehicle-, control LNP- and Triplet LNP-treated mice. One-way ANOVA followed by post hoc Tukey’s multiple comparisons test was performed. E UMAP plot shows high-resolution clustering of the neutrophil compartment in the tumor CITE-seq dataset. Bars show the relative abundance of the different neutrophil subsets in the tumor CITE-seq dataset across different treatments. F Circle plot shows links between top predicted ligands for migDC, cDC2, cDC2 prolif, Mac Folr2, Mac MHC-II-hi, Mac, Mono, Neutro IFN, and the Neutro clusters and their associated receptors found on CD8⁺ T cells within the control niche and Triplet LNP niche of the tumor CITE-seq dataset. The transparency of the linking arrow reflects the prioritization score of the ligand-receptor pair. The heatmaps below show the scaled average expression level of the top target genes that are potentially regulated by the predicted ligand-receptor pairs. G Schematic outline of experimental procedures. H MC38 tumor growth curves upon isotype/control diet or αLy6G or PLX5622 diet administration (n = 6 animals/group). Repeated measures two-way ANOVA followed by post hoc Tukey’s multiple comparisons test were performed. Error bars indicate SEM. Source data for (C, D, H) are provided as a Source Data file. CITE, cellular indexing of transcriptomes and epitopes by sequencing; SEM, standard error of mean; UMAP, uniform manifold approximation and projection.

Full size image

Triplet LNP enhances CD8⁺ T-cell activation and proliferation in the tdLN

In contrast to the TME, within the tdLN of MC38 tumor-bearing mice, the Triplet LNP treatment had a less profound effect on the relative abundance of the immune cell populations two days after the second intratumoral injection (Fig. 3A). We observed a slight increase in B cell frequency, accompanied by a decrease in the frequency of CD4⁺ T cells, including FoxP3⁺ Tregs (Fig. 6A and Supplementary Fig. 12A–C). While the Triplet LNP had no impact on the frequency of CD8⁺ T cells in the tdLN, it induced CD8⁺ T-cell proliferation and enhanced their cytotoxic capacity and their differentiation toward a central memory phenotype (Fig. 6B–D and Supplementary Fig. 12D). Of note, CD8⁺ T-cell activation was also observed in the tdLN of Triplet LNP-treated E0771 tumor-bearing mice (Supplementary Fig. 12E, F). Next, we performed CITE-seq on CD45⁺ cells of tdLN of vehicle-, control LNP- and Triplet LNP-treated mice (Fig. 6E). High-resolution multimodal clustering of the T/NK-cell compartment revealed thirteen distinct clusters, of which the abundance of CD8T Ctla2a Ifng, CD8T Ctla2a and CD8T prolif clusters was slightly increased in the tdLN of the Triplet LNP-treated mice (Fig. 6E and Supplementary Fig. 12G). The Triplet LNP treatment induced the expression of various effector genes in the CD8T Ctla2a Ifng cluster, including Prf1, Ifng, Gzma, Gzmb, Fasl, and Tnf. Interestingly, it also induced the expression of the chemokine receptor Cxcr6, which has been reported to play a crucial role in the optimal positioning of CD8⁺ T cells to interact with activated DCs within the TME⁴⁵ (Fig. 6F). Since our data suggests that LNP uptake might promote cDC2 activation and migration toward the tdLN (Fig. 1E–G), we sought to determine the effect of the Triplet LNP treatment on DC-CD8⁺ T-cell interactions in the tdLN. For this, we performed a differential Nichenet analysis to predict niche-specific ligand-receptor pairs between different cDC subsets and CD8⁺ T cells in the control niche (Vehicle + Control LNP) and the Triplet LNP niche (Supplementary Fig. 12H, I). The analysis predicted different interactions in the Triplet LNP niche, in particular, Triplet LNP induced migDC-CD8⁺ T-cell and cDC2-CD8⁺ T-cell interaction pairs in which Cd80, Cd86, Cxcl16, and Cxcl10 were among the top differentially expressed ligands (Supplementary Fig. 12I). The target genes of these ligand-receptor interactions were mapped, highlighting the upregulated expression of genes associated with CD8⁺ T-cell effector function and proliferation in the Triplet LNP niche, including Ccl4, Ccl5, Gzma, Ifng, and Mki67 (Supplementary Fig. 12I). Hence, this data suggests that upon Triplet LNP treatment, cDC2s and migDCs induce cytotoxic CD8⁺ T cells in the tdLNs. To investigate whether the trafficking of the CD8⁺ T cells from the tdLN to the tumor is necessary for the therapeutic efficacy of the Triplet LNP, we blocked T-cell egression using the S1PR inhibitor FTY720 (Supplementary Fig. 12J). FTY720 treatment diminished the therapeutic efficacy of the Triplet LNP (5 of 11 complete regressors), indicating that CD8⁺ T-cell trafficking is necessary for optimal therapeutic efficacy (Fig. 6G). Given that Triplet LNP therapy induces central memory CD8⁺ T cells in the tdLN, we investigated whether the Triplet LNP treatment conferred systemic long-term immunity. Indeed, we found that splenic CD8⁺ T cells from Triplet LNP-treated mice secreted higher levels of IFN-γ in response to the p15E peptide, an immunodominant MHC-I-restricted epitope of MC38 (Fig. 6H). More importantly, Triplet LNP-treated complete regressors remained immune to MC38 and E0771 cancer cells when rechallenged sixty days after initial tumor inoculation (Fig. 6I).

A Pie charts show the mean frequency of immune subsets within CD45⁺ cells in the tdLN quantified by flow cytometry (n = 7 animals/group). Bar graphs show the frequency of (B) Ki-67⁺ CD8⁺ T cells, (C) Granzyme B⁺ CD8⁺ T cells and (D) CD44^hi CD62L^hi CD8⁺ T cells in the tdLN (n = 7 animals/group). E UMAP plot of CITE-seq dataset containing CD45⁺ sorted cells from tdLN of vehicle, control LNP, and Triplet LNP-treated mice (n = 5 animals/group). High-resolution clustering of the T-cell compartment of the dataset is shown on the right. Below, pie charts show the relative abundance of different T-cell subsets in the tdLN dataset across different treatments. F Dot plot highlights differentially expressed genes associated with effector function and migration in distinct CD8⁺ T-cell clusters of the tdLN CITE-seq dataset across different treatments. Dot size represents the percentage of cells expressing the gene and color gradient represents average scaled expression within a cell cluster. G Schematic outline of experimental procedures and survival curves of MC38 tumor-bearing mice treated with vehicle, control LNP or Triplet LNP in the presence or absence of FTY720 (0.9% NaCl/vehicle n = 12 animals; 0.9% NaCl/control LNP n = 10 animals; 0.9% NaCl/triplet LNP n = 11 animals; FTY720/vehicle n = 11 animals; FTY720/control LNP n = 12 animals; FTY720/triplet LNP n = 11 animals). H Bar graph shows the IFN-γ-secreting CD8⁺ T cells from spleens of naïve mice (n = 8 animals), vehicle-treated mice (n = 7 animals), control LNP-treated mice (n = 6 animals), and Triplet LNP-treated mice (n = 8 animals). I Schematic outline of experimental procedures and MC38 (control n = 17 animals; rechallenge=8 animals) and E0771 (control n = 4 animals; rechallenge n = 6 animals) tumor growth curves of control mice and rechallenged complete regressors. B–D, H One-way ANOVA followed by post hoc Tukey’s multiple comparisons test and (G) Gehan-Breslow-Wilcoxon test were performed. Data shown in (A–D) are representative of three independent experiments. Data shown in (G) is pooled from two independent experiments. Bars show mean. Error bars indicate SEM. Source data for (A–D, G–I) are provided as a Source Data file. CITE, cellular indexing of transcriptomes and epitopes by sequencing; FTY720, fingolimod; tdLN, tumor-draining lymph node; SEM, standard error of mean; UMAP, uniform manifold approximation and projection.

Full size image

Intratumoral injection of Triplet LNP induces regression of distal tumors

Having demonstrated that the Triplet LNP treatment led to CD8⁺ T-cell activation in the tdLN, conferred long-term immunity and significantly increased the frequency of tumor-matching CD39⁺ CX3CR1⁺ CD8⁺ T cells in the blood, we aimed at evaluating the abscopal effects of the Triplet LNP treatment. Hereto, mice were inoculated with MC38 tumors at two different subcutaneous sites (left and right flanks), but only the tumor on the right flank was treated with vehicle, control LNP or Triplet LNP (Fig. 7A). While the Triplet LNP-treated tumors all completely regressed, interestingly, the treatment significantly reduced the growth of the distal nontreated tumor, improving the overall survival (Fig. 7B–D). Similarly, in the less immunogenic E0771 model (Fig. 7E), the Triplet LNP therapy also reduced local and distal tumor growth, prolonging overall survival (Fig. 7F–H). In addition, we utilized a pseudometastasis approach in which mice were inoculated with E0771 cancer cells in the mammary fat pad before they received an intravenous injection of E0771 cancer cells a day later to induce lung metastasis (Supplementary Fig. 13A). Local injection of the Triplet LNP reduced lung tumor burden and improved overall survival, leading to 2 out of 6 complete regressors (Supplementary Fig. 13B–D).

A Schematic outline of experimental procedures. Created in BioRender. Laoui, D. (2024) https://BioRender.com/j25s690. B MC38 tumor individual growth curves of treated and nontreated tumors. Treated (right flank) tumors received three injections of vehicle, control LNP or Triplet LNP (n = 12 animals/group). Vertical dotted lines indicate intratumoral injections of the treated tumors. C MC38 tumor growth curves of treated and nontreated tumors (n = 12 animals/group). Vertical dotted lines indicate intratumoral injections of the treated tumors. D Survival curves of MC38 tumor-bearing mice treated with vehicle, control LNP or Triplet LNP (n = 12 animal/group). E Schematic outline of experimental procedures. Created in BioRender. Laoui, D. (2024) https://BioRender.com/l86l838F E0771 tumor individual growth curves of treated and nontreated tumors. Treated (right flank) tumors received three injections of vehicle, control LNP or Triplet LNP (n = 6 animals/group). Vertical dotted lines indicate intratumoral injections of the treated tumors. G E0771 tumor growth curves of treated and nontreated tumors (n = 6 animals/group). Vertical dotted lines indicate intratumoral injections of the treated tumors. H Survival curves of E0771 tumor-bearing mice treated with vehicle, control LNP or Triplet LNP (n = 6 animals/group). C, G Repeated measures two-way ANOVA followed by post hoc Tukey’s multiple comparisons test and (D, H) log-rank (Mantel-Cox) test were performed. Horizontal lines show mean. Data shown in (B–D) are pooled from two independent experiments. Error bars indicate SEM. Source data are provided as a Source Data file. SEM, standard error of mean.

Full size image

LNP-formulated mRNA leads to efficient protein expression in human cells

To assess the capacity of the LNPs to transfect human cells, we treated single-cell suspensions of tumor biopsies acquired from patients with NSCLC with vehicle, Thy1.1 mRNA or LNP-formulated Thy1.1 mRNA and quantified Thy1.1 expression 18 h later by flow cytometry using the gating strategies depicted in Supplementary Fig. 14A, B. Thy1.1 LNPs resulted in improved expression compared to naked Thy1.1 mRNA (Fig. 8A). The Thy1.1 LNP transfected both the CD45⁺ immune cells and CD45^– non-immune cells (Fig. 8B). Within the CD45⁺ compartment, Thy1.1 expression was most pronounced in myeloid cells, in particular, macrophages (Fig. 8C–E). This data indicates that the LNPs can efficiently transfect immune and non-immune human cells with a noticeable bias toward myeloid cells. Finally, we sought to corelate the expression of IL21, IL7, and TNFSF9 with the overall survival of patients with cancer that were not treated with immunotherapy, including 9 tumor types, using the KM plotter⁴⁶. This analysis demonstrates that higher expression of IL21, IL7, TNFSF9, and their combination are associated with better overall survival with the expression of the combination showing the most significant correlation with better overall survival (Fig. 8F), suggesting that the Triplet LNP might potentially improve the survival of patients with cancer.

A Representative flow cytometry plots and bar graph show the frequency of Thy1.1⁺ cells in cell suspensions of NSCLC biopsies 18 h post incubation with vehicle, 500 ng/ml naked Thy1.1 mRNA, or 500 ng/ml Thy1.1 LNP (n = 3 NSCLC biopsies/group). One-way ANOVA followed by post hoc Tukey’s multiple comparisons test was performed. B Scatter plot shows the frequency of CD45⁺ and CD45^– cells within Thy1.1⁺ cells (n = 3 NSCLC biopsies). C Representative flow cytometry plots show the frequency of Thy1.1⁺ cells within myeloid and lymphoid lineages. D Scatter plot shows the frequency of myeloid and lymphoid cells within Thy1.1⁺ CD45⁺ cells (n = 3 NSCLC biopsies). E Bar graph shows the frequency of immune cell subsets within Thy1.1⁺ CD45⁺ cells (n = 3 NSCLC biopsies). F Kaplan-Meier estimates of overall survival comparing the top (high) and bottom (low) of cancer patients based on the median expression of the indicated genes. Hazard ratio (HR) with 95% confidence interval. p values calculated using log rank test. Tick marks indicate censoring. Bars and horizontal lines show mean. Error bars indicate SEM. Source data for (A, B, D, E) are provided as a Source Data file. NSCLC, non-small cell lung carcinoma; SEM, standard error of mean.

Full size image

• SEO Powered Content & PR Distribution. Get Amplified Today.
• PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
• PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
• PlatoESG. Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
• PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
• Source: https://www.nature.com/articles/s41467-024-54877-9