Abstract
Lipid nanoparticles (LNPs) can be designed to potentiate cancer immunotherapy by promoting their uptake by antigen-presenting cells, stimulating the maturation of these cells and modulating the activity of adjuvants. Here we report an LNP-screening method for the optimization of the type of helper lipid and of lipid-component ratios to enhance the delivery of tumour-antigen-encoding mRNA to dendritic cells and their immune-activation profile towards enhanced antitumour activity. The method involves screening for LNPs that enhance the maturation of bone-marrow-derived dendritic cells and antigen presentation in vitro, followed by assessing immune activation and tumour-growth suppression in a mouse model of melanoma after subcutaneous or intramuscular delivery of the LNPs. We found that the most potent antitumour activity, especially when combined with immune checkpoint inhibitors, resulted from a coordinated attack by T cells and NK cells, triggered by LNPs that elicited strong immune activity in both type-1 and type-2 T helper cells. Our findings highlight the importance of optimizing the LNP composition of mRNA-based cancer vaccines to tailor antigen-specific immune-activation profiles.
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Data availability
The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data for the figures are provided with this paper.
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Acknowledgements
S.C.M. and H.-Q.M. disclose support for the research described in this study from the National Institutes of Health (U01AI155313). J.P.S., J.J.G. and H.-Q.M. also disclose support for the publication of this study from the National Institutes of Health (P41EB028239).
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Y.Z. and H.-Q.M. conceived and designed the study. H.-Q.M. and S.C.M. secured the funding for the study. Y.Z., J.M., R.S., Z.-C.Y., I.V., J.L., X.L., L.C., W.H.T., N.M.K., C.W., W.J.C. and J.K. performed the experiments. Y.Z., J.M., R.S., J.L.S., I.V., Y.H., W.J.C., R.A.R., M.J.S., N.K.L., S.L., G.P.H., S.K.R., S.Y.T., D.J.Z., J.J.G., L.Z., J.C., J.P.S., S.C.M. and H.-Q.M. participated in data analysis and interpretation. The manuscript was written by Y.Z. and H.-Q.M., with revisions made by S.C.M., C.W., R.S., I.V., S.Y.T., W.J.C., J.C.D., L.Z., J.P.S., R.A.R., S.K.R. and J.J.G., and with input from all the other authors.
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H.-Q.M., Y.Z., J.M, R.S, L.C., I.V. and S.K.R. are co-inventors of a pending patent application (PCT/US2023/016938, filed in March 2023) covering the LNP formulation described in this paper. The patent was filed through Johns Hopkins Technology Ventures and is managed by it. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Secretion levels of cytokines within the supernatant of BMDCs after 24 h of incubation.
Secretion levels of IL-6 (a), TNF-α (b) and IFN-γ (c), within the supernatant of BMDCs after 24 h incubation with the three mOVA-loaded LNPs were measured by ELISA. Data are represented as the mean ± s.e.m. Data were analyzed using one-way ANOVA and Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant; BMDC, bone marrow derived dendritic cell; ELISA, enzyme-linked immunoassay.
Extended Data Fig. 2 IL-6 and TNF-α secretion levels from antigen-stimulated splenocytes.
Splenocytes were isolated from vaccinated mice and restimulated in vitro with OVA and SIINFEKL peptide (100 μg ml−1 OVA and 2 μg ml−1 SIINFEKL) for 72 h. Secretion levels of IL-6 (a) and TNF-α (b) within the supernatant of were measured by ELISA. ‘Algel+OVA’ stands for Alhydrogel®+OVA group. Data represent the mean ± s.e.m. from a representative experiment (n = 4 biologically independent samples) of two independent experiments. Data were analyzed using one-way ANOVA and Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant; BMDC, bone marrow derived dendritic cell; ELISA, enzyme-linked immunoassay.
Extended Data Fig. 3 Titres of OVA-specific IgG subclass antibody in blood-serum samples collected on day 21 following immunization.
IgG1 (a) and IgG2a (b) antibodies in blood serum on day 21 were determined by ELISA. ‘Algel+OVA’ stands for Alhydrogel®+OVA group. Data represent the mean ± s.e.m. from a representative experiment (n = 4 biologically independent samples) of two independent experiments. Data were analyzed using one-way ANOVA and Dunnett’s multiple comparisons test. ****P < 0.0001; ELISA, enzyme-linked immunoassay.
Extended Data Fig. 4 Cytokine levels in the local injection site measured at 4 h or 24 h post-administration of the top-performing LNPs.
ELISA was employed to quantify the cytokine levels, including IFN-γ (a), TNF-α (b), and IL-4 (c), at the local injection site using OVA-encoding mRNA after the administration of three formulations (C10, D6, and F5) at 4 h and 24 h. The local injection sites were collected, homogenized, and subjected to tissue lysis to extract the proteins. The resulting lysate was centrifuged to separate the insoluble cellular debris. The protein concentration in each sample was determined using the BCA assay and normalized accordingly. Data represent the mean ± s.e.m. with n = 4 biologically independent samples. Data were analyzed using one-way ANOVA and Tukey’s multiple comparisons test. *P < 0.05; ***P < 0.001; NS, not significant.
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Zhu, Y., Ma, J., Shen, R. et al. Screening for lipid nanoparticles that modulate the immune activity of helper T cells towards enhanced antitumour activity. Nat. Biomed. Eng (2023). https://doi.org/10.1038/s41551-023-01131-0
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DOI: https://doi.org/10.1038/s41551-023-01131-0