LNP-RNA-mediated antigen presentation leverages SARS-CoV-2-specific immunity for cancer treatment

Abstract

Lipid nanoparticle (LNP)-mRNA vaccines have demonstrated protective capability in combating SARS-CoV-2. Their extensive deployment across the global population leads to the broad presence of T-cell immunity against the SARS-CoV-2 spike protein, presenting an opportunity to harness this immunological response as a universal antigen target for cancer treatment. Herein, we design and synthesize a series of amino alcohol- or amino acid-derived ionizable lipids (AA lipids) and develop an LNP-RNA-based antigen presentation platform to redirect spike-specific T-cell immunity against cancer in mouse models. First, in a prime-boost regimen, AA2 LNP encapsulating spike mRNA elicit stronger T-cell immunity against the spike epitopes compared to FDA-approved LNPs (ALC-0315 and SM-102), highlighting the superior delivery efficiency of AA2 LNP. Next, AA15V LNP efficiently delivers self-amplifying RNAs (saRNAs) encoding spike epitope-loaded single-chain trimer (sSE-SCT) MHC I molecules into tumor tissues, thereby inducing the presentation of spike epitopes. Our results show that a single intratumoral (i.t.) treatment of AA15V LNP-sSE-SCTs suppresses tumor growth and extends the survival of B16F10 melanoma and A20 lymphoma tumor-bearing mice vaccinated with AA2 LNP-spike mRNA. Additionally, AA15V LNP-sSE-SCTs enable SE-SCT expression in ex vivo human glioblastoma and lung cancer samples, suggesting its potential in clinical translation.

Fig. 1. LNP-RNA-mediated antigen presentation leverages SARS-CoV-2-specific T-cell immunity to treat tumors.

a Illustration of LNP-RNA-mediated antigen presentation platform. E spike epitope, TM transmembrane domain. Created in BioRender. Xue, Y. (2025) https://BioRender.com/g18v799. b Design and structures of AA lipids. c General synthetic routes of AA lipids.

Fig. 2. Investigation of AA LNPs for spike mRNA vaccination.

a The luminescence intensity in C2C12 cells. b The luminescence intensity in JAWSII cells. c The luminescence intensity of the muscle in the lead AA LNP-FLuc mRNA-treated C57BL/6 mice. The intensity was normalized to the ALC-0315 LNP group. d Representative images of C57BL/6 mice i.m. treated with AA2 LNP-FLuc mRNA, ALC-0315 LNP-FLuc mRNA, and SM-102 LNP-FLuc mRNA. e Spike-specific IgG titer in blood drawn from C57BL/6 mice vaccinated with AA2 LNP-spike mRNA, ALC-0315 LNP-spike mRNA or SM-102 LNP-spike mRNA. Data in (a–d) are from n = 3 biologically independent samples. Data in (e) are from n = 6 biologically independent samples. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 3. AA2 LNP-spike mRNA vaccination generates spike-specific T-cell immunity.

a Schematic depicting activation-induced marker (AIM) assay of T cells isolated from spleen and blood of mice vaccinated with AA2 LNP-spike mRNA or SM-102 LNP-spike mRNA. Created in BioRender. Xue, Y. (2025) https://BioRender.com/z43s143. b, d Spike epitope-specific activation of CD4⁺ T cells isolated from the blood (b) and spleen (d) of the mice vaccinated with AA2 LNP-spike mRNA or SM-102 LNP-spike mRNA. CE control epitope, SE spike epitope. c, e Spike epitope-specific activation of CD8⁺ T cells isolated from the blood (c) and spleen (e) of the mice vaccinated with AA2 LNP-spike mRNA or SM-102 LNP-spike mRNA. Gzmb granzyme B, CE control epitope, SE spike epitope. Data are from n = 5 biologically independent samples and are presented as mean ± SD. Statistical significance was determined by the two-tailed Student’s t-test. n.s. not significant P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4. Treatment with AA15V LNP-sSE-SCTs enhances spike-specific T cell-mediated killing of cancer cells.

a Screening of AA LNPs in B16F10 cells. b Construction of saRNA encoding MHC I single-chain trimer (SCT) containing spike epitopes. E spike epitope, TM transmembrane domain. Created in BioRender. Xue, Y. (2025) https://BioRender.com/k91i086. c Dynamic expression of H-2Kb⁺β2m⁺ in B16F10 cells. d Expression of OVA-H-2Kb⁺ in B16F10 cells after a single i.t. injection of AA15V LNP-sOP-SCTs. OP, OVA_257-264 peptide (SIINFEKL). e Percentage of apoptotic B16F10 cells in the T cell-mediated killing assay. f Percentage of dead B16F10 cells in the T cell-mediated killing assay. g, h Spike epitope-specific activation of CD4⁺ T cells (g) and CD8⁺ T cells (h) isolated from B16F10 tumors in mice vaccinated with AA2 LNP-spike mRNA. CE control epitope, SE spike epitope. Data in (a) and (c) are from n = 3 biologically independent samples. Data in (d) are from n = 4 biologically independent samples. Data in (e–h) are from n = 5 biologically independent samples. Data are presented as mean ± SD. Statistical significance in (a, c) and (e, f) was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. Statistical significance in (d) and (g, h) was determined by the two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5. AA15V LNP-sSE-SCTs redirects spike-specific T-cell immunity to treat tumors and reprogram TME.

a Schematic of the treatment regimen in the B16F10 tumor model. Created in BioRender. Xue, Y. (2025) https://BioRender.com/b49g753. b Tumor volumes in different groups. c Survival rates of the mice in the B16F10 tumor model. d Expression of cytokines and chemokines in tumor samples at 24 h post-treatment. e Expression level of representative cytokines and chemokines in tumor samples from (d). f Expression of cytokines and chemokines in blood samples at 24 h post-treatment. g Expression level of representative cytokines and chemokines in blood samples from (f). h–l Immune cell populations in tumor tissues. Populations of i activated DCs, j activated macrophages, k primed CD4⁺ T cells, and l primed CD8⁺ T cells in tumor tissues. Data in (b, c) and (h–l) are from n = 5 biologically independent samples. Data in (d–g) are from n = 3 biologically independent samples. Data are presented as mean ± SD. Statistical significance in (b) was determined by two-tailed Student’s t-test. Statistical significance in (c) was determined by the log-rank (Mantel–Cox) test. Statistical significance in (e–l) was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. n.s. not significant P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6. Applicability of AA15V LNP-sSE-SCTs in multiple tumor models and human tumor samples.

a Schematic of the treatment regimen in B16F10 tumor model treated with the combination of AA15V LNP-sSE-SCTs and ICI. b Tumor volumes over time. c Survival rates of mice bearing B16F10 tumor. d Schematic of the treatment regimen in A20 tumor model. e Tumor volumes over time. f Survival rates of the mice bearing A20 tumor. g Schematic depicting ex vivo AA15V LNP-sSE-SCTs delivery in human tumor tissues. Created in BioRender. Xue, Y. (2025) https://BioRender.com/q02v567. h Expression of H-2Kb⁺β2m⁺ expression in CD45⁻ cells from pediatric glioma dissections after ex vivo treatment with AA15V LNP-sSE-SCTs. i, j H-2Kb⁺β2m⁺ in CD45⁻ cells from two separate lung left lower lobe (LLL) adenocarcinoma specimens after ex vivo treatment with AA15V LNP-sSE-SCTs. Data in (b, c) are from n = 6 (PBS group) and 8 (SCT groups) biologically independent samples, respectively. Data in (e, f) are from n = 6 (PBS group and UnVac groups) and 7 (Vac groups) biologically independent samples, respectively. Data in (h) and (j) are from n = 3 individual tissue slices. Data in (i) are from n = 4 individual tissue slices. Data are presented as mean ± SD. Statistical significance in (b, e) and (h–j) was determined by the two-tailed Student’s t-test. Statistical significance in (c) and (f) was determined by the log-rank (Mantel–Cox) test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.