Small circular RNAs as vaccines for cancer immunotherapy

Abstract

Messenger RNA vaccines have shown strong prophylactic efficacy against viral infections. Here we show that antigen-encoding small circular RNAs (circRNAs) loaded in lipid nanoparticles elicit potent and durable T cell responses for robust tumour immunotherapy after subcutaneous injection in mice, particularly when combined with immune checkpoint inhibition. The small circRNA vaccines are highly stable and show low levels of activation of protein kinase R as well as low cytotoxicity, enabling long-lasting antigen translation (longer than 1 week in cells). Relative to large protein-encoding unmodified or modified mRNAs and circRNAs, small circRNA vaccines elicited up to 10-fold antigen-specific T cells in mice and accounted for 30-75% of the total peripheral CD8⁺ T cells over 6 months. Small circRNA vaccines encoding tumour-associated antigens, neoantigens and oncoviral or viral antigens elicited substantial CD8⁺ and CD4⁺ T cell responses in young adult mice and in immunosenescent aged mice. Combined with immune checkpoint inhibition, monovalent and multivalent circRNA vaccines reduced tumour-induced immunosuppression and inhibited poorly immunogenic mouse tumours, including melanoma resistant to immune checkpoint blockade.

Extended Data Fig. 1 ∣. Liposomes promote circRNA delivery and antigen presentation in DCs.

a, DLS data showing the size distribution of blank liposome and lipo-circRNA with different N/P ratios in PBS (solid) and 1% FBS (dotted). b, Zeta potential of blank liposome and lipo-circRNA. c-d, In vitro cell uptake of circRNA and antigen presentation in DCs mediated by lipo-circRNA. c, Flow cytometry results and d, MFI of the SIINFEKL presentation in SIINFEKL-circRNA-treated DC2.4 cells (24 h). SIINFEKL antigen presentation on DC2.4 cells that were treated with Lipofectamine 3000-transfected circRNA and lipo-circRNA with different N:P ratios, respectively. Ns: non-significant, *p < 0.05, ***p < 0.001, by one-way ANOVA with Bonferroni post-test. e-h, In vitro intracellular delivery of circRNA in DCs by liposome. e, Confocal microscopy images of DC2.4 cells treated with lipo-circRNA for 1 h, 3 h, or 6 h. Blue: nuclei stained with Hoechst33342. Green: endolysosome stained with LysoTracker Green DND-26. Red: Cy5-circRNA. Insets: close-up views of single cells. f, Flow cytometry results of DC2.4 cells incubated with Lipo-circRNA and free circRNA for different times. g, Flow cytometry results showing MFI of DCs incubated with free or Lipo-Cy5-circRNA. ****p < 0.0001, by t-test of the AUC. h, The signal ratio of Cy5-circRNA outside/inside (O/I) the endolysosome.

Extended Data Fig. 2 ∣. Liposomes promoted the delivery of circRNA to draining lymph nodes and to key intranodal APC subsets in mice.

a, Liposomes promoted the delivery of IR800-circRNA to draining popliteal lymph nodes (circled) in BALB/c mice (0.2 nmol, s.c. injected at foot pad). b, AUC of the radiance efficiency. **p < 0.01, by t-test of the AUC. c, Ex vivo fluorescence images of BALB/c mice after s.c. injected at tail base for 24 h. d, Radiance efficiency of major organs and inguinal lymph nodes after s.c. injection of IR800-circRNA at tail base for 24 h. He: heart; Li: liver; Sp: spleen; Lu: lung; Ki: kidney; and LN: inguinal lymph node. e, The frequency of circRNA⁺ cDCs and macrophages in inguinal lymph node tissues 24 h after s.c. injection at tail base.

Extended Data Fig. 3 ∣. Small circRNA-SIINFEKL elicited potent T-cell responses in young adult mice.

a, Study design. C57BL/6 mice (n = 5) were vaccinated on day 0 and day 14, and PBMC T cell responses were analyzed starting from day 21. b, Tetramer staining on day21 – day70 showed that circRNA-SIINFEKL elicited potent SIINFEKL-specific CD8⁺ T cell response in mice (n = 5) that outperformed current benchmarks 5moU-modified CleanCap mRNA-OVA and CpG-adjuvanted OVA. c, circRNA-SIINFEKL upregulated PD-1 expression on SIINFEKL-specific CD8⁺ T cells on day21. d, circRNA-SIINFEKL elicited SIINFEKL-specific T cell memory (day 70). Tem: effector memory T cell; Tcm: central memory T cell. e, PD-1 MFI on live PBMC CD8⁺ T cells. f, circRNA-SIINFEKL enhanced PD-1 expression levels (MFI) and frequencies on SIINFEKL⁺CD8⁺ T cells than that on total CD8⁺ T cells in peripheral blood on day 21, indicating immune exhaustion often resulting from chronic immunostimulation and providing an opportunity to combine circRNA vaccines with immune checkpoint blockade for optimal T cell responses (Two-tailed paired t test). g, circRNA-SIINFEKL enabled mice to resist 3×10⁵ EG7.OVA tumour challenge 70 days post-vaccination. Vaccine delivery by liposome, s.c. injected at tail base, 5 μg RNA, 2 nmole CpG, 20 μg OVA. *: relative to circRNA. h, Mouse body weights after tumour challenge. Data represent mean ± SD (b-e) and mean ± s.e.m. in other figure panels (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by one-way ANOVA with Bonferroni post-test unless denoted otherwise.

Extended Data Fig. 4 ∣. Peptide-encoding small circRNA vaccine elicit stronger and more durable T-cell responses than protein-encoding unmodified mRNA and large circRNA vaccines in young adult mice.

a, Design of T cell response study in mice. C57BL/6 mice (n = 4-5; 6-8 weeks) were immunized with circRNA-SIINFEKL, unmodified mRNA-OVA, and large circRNA code OVA protein at 3 μg and 10 μg doses, respectively. b, 120-day kinetics of the PBMC SIINFEKL-specific CD8⁺ T cell percentages in the above immunized mice, suggesting that circRNA-SIINFEKL elicited overall stronger and more durable T cell responses than OVA-encoding unmodified mRNA and large circRNA vaccines. Asterisks: statistical significance of the T cell fraction AUC relative to that for circRNA. c, circRNA-SIINFEKL elicited larger fractions of memory T cells (Tem + Tcm) than unmodified mRNAs (day 90), indicating great T cell memory elicited by small circRNA vaccine. Data were quantified from CD44 and CD62L staining of PBMC CD8⁺ T cells.

Extended Data Fig. 5 ∣. Monovalent small circRNA vs modified mRNA or large circRNA vaccines for robust tumour immunotherapy.

a, For immunotherapy studies in mouse models of subcutaneous EG7.OVA (b-d), and TC-1 (e), tumours were inoculated into the right flank of C57Bl/6 mice. Vaccine: 5 μg RNA or 5 μg CpG + 10 μg protein or peptide antigens, s.c. injected in liposome at mouse tail base; antibodies: 200 μg, i.p. b-d, EG7.OVA tumour growth (b) and Kaplan-Meier survival curves (c) of EG7.OVA tumour-bearing mice treated with circRNA-SIINFEKL, 5moU-mRNA-OVA, and CpG-adjuvanted OVA, respectively. αCD8, αCD4, and αNK1.1 were injected intraperitoneally (i.p.) for lymphocyte depletion. d, Body weights of EG7.OVA tumour-bearing mice treated with circRNA-SIINFEKL vs. controls. e, Individual (upper panel) and average (lower panel) EG7.OVA tumor growth curves in C57BL/6 mice treated with the indicated circRNA-SIINFEKL or large circRNA-OVA, as well as αPD-1 (i.p.) alone or combined with circRNA-SIINFEKL. RNA: 30 μg, s.c. injection at tail base. CR: complete regression rate. f, TC-1 tumour volumes after lymphocyte depletion using αCD8, αCD4 or αNK1.1. Data represent mean ± s.e.m. *p < 0.05; **p < 0.01; ***p < 0.001, by one-way ANOVA with a Bonferroni post-test (n = 6-8).

Fig. 1 ∣. Schematic illustration of highly stable small circRNA vaccines that elicit potent and long-lasting T cell responses for tumour immunotherapy.

Small circRNA consists of peptide-antigen-encoding RNA and a short IRES and a Kozak consensus sequence that initiate peptide translation. Small circRNA was synthesized by ligating RNA oligonucleotide precursor(s) using RNA ligase and DNA splint(s). The absence of termini and the minimal sizes of small circRNA enable it to resist exonuclease degradation and minimize endonuclease degradation and hydrolysis, resulting in excellent thermostability and biostability. Nanocarriers delivered small circRNA vaccines to draining lymph nodes and intranodal APCs, in which (1) circRNA activates PRRs to elicit pro-inflammatory innate immunity with low PKR activation and low cytotoxicity, and (2) circRNA is efficiently translated to antigen peptides for antigen presentation over a prolonged duration. This allows small circRNA vaccines to elicit potent and long-lasting antigen-specific T cell responses. Modular small circRNA vaccines can be easily adjusted to encode various peptide antigens for versatile applications: (1) by encoding MHC-I- and MHC-II-restricted antigens, circRNA elicits CD8⁺ and CD4⁺ T cell responses, respectively; and (2) by encoding tumour-associated antigens, tumour neoantigens or (onco)viral antigens, circRNA holds the potential to develop off-the-shelf shared vaccines and personalized vaccines. Moreover, small circRNA vaccines reduce the immunosuppression and enhance the infiltration of antitumour immune cells in distant tumours. As a result, small circRNA vaccines, especially when combined with ICB, show robust immunotherapeutic efficacy for multiple types of tumour, including ICB-resistant Braf^V600E melanoma.

Fig. 2 ∣. Highly stable small circRNA vaccines for efficient peptide translation and antigen presentation.

a, Top: schematic illustration of the synthesis of small circRNA and liRNA by ligation of RNA oligonucleotide precursors and DNA splints. Bottom: Sanger sequencing of the cDNA of circRNA-SIINFEKL indicates precise and uniform ligation of RNA precursors into circRNA. The denoted RNA sequence is converted from the Sanger sequencing results of cDNA. b, crTMV IRES-based circRNA-SIINFEKL elicited efficient antigen presentation and T cell priming. Left: flow cytometric quantification of the mean fluorescence intensity (MFI) of SIINFEKL/H-2K^b complexes on DC2.4 cells treated with circRNA or controls for 24 h. Right: the activities of SIINFEKL-specific B3Z CD8⁺ T cell hybridoma co-incubated with the as-treated DC2.4 cells. B3Z cell activity: absorbance value. c, The secondary structures of crTMV IRES (left) and crTMV-based circRNA-SIINFEKL (right), as predicted using NUPACK. The blue box denotes IRES. d, Potent and durable priming of B3Z T cells by DC2.4 cells treated with circRNA-SIINFEKL. 5moU-mRNA-OVA: a benchmark protein-encoding 5moU-modified mRNA with CleanCap, 5′- and 3′-UTRs, and 3′ A120. e, Confocal fluorescence microscopy images show the efficient production of a model peptide, Flag, from circRNA-Flag in live DC2.4 cells 24 h after transfection. f–h, circBroccoli was highly stable in live DC2.4 cells as shown by flow cytometry (f,g) and confocal fluorescence microscopy (h) analysis of cell Broccoli fluorescence intensities. Cells were transfected with circBroccoli or linear Broccoli control for 1–168 h, before adding fluorescence-activating Broccoli cognate, DFHBI-1T. Asterisks in g denote statistical significance of Broccoli MFI AUC of linear Broccoli relative to that of circBroccoli. i, circRNA-seq results showing the sequence integrity of circRNA-SIINFEKL recovered from live DCs after transfection for 24 h. circRNA-SIINFEKL in PBS was used as a positive control. The authenticity rate of each nucleotide was calculated as the rate of unmutated nucleotides. j, Percentages of intact circRNA-SIINFEKL, liRNA-SIINFEKL and 5moU-mRNA-OVA after storage in PBS at −20 °C, 4 °C or 23 °C for up to 180 days. At −20 °C, 4 °C and 23 °C, the half-lives of circRNA-SIINFEKL are estimated to be 401, 78 and 16 days, respectively; 143, 44 and 6 days, respectively, for 5moU-mRNA-OVA; and 2.6, <0.5 and <0.5 days, respectively, for liRNA-SIINFEKL. Data were quantified from gel electrophoresis using ImageJ. k, Upon transfection into DC2.4 cells, small circRNA-Flag showed durable Flag expression for at least 7 days, in contrast to fLuc expression for less than 3 days from 5moU-mRNA-fLuc. Intracellular Flag was stained using phycoerythrin (PE)-conjugated anti-Flag antibody, and the MFI of cells was measured by flow cytometry. l, Dynamic light scattering (left) and cryogenic electron microscopy (cryo-EM) images (right) showing the sizes and morphology of blank and circRNA-loaded ionizable SM-102 LNPs. m, Percentages of intact circBroccoli loaded in LNPs after storage in PBS at 4 °C or −20 °C (8% cryoprotectant sucrose) for 72 days. Data were quantified from flow cytometric analysis of DC2.4 cells transfected (24 h) with circBroccoli LNPs recovered from storage (paired t-test). n,o, Representative agarose gel electrophoresis images (n) and the intact circRNA percentages (o) of circRNA-SIINFEKL (1 nmol) and circRNA-RBD (1 nmol) after incubation in a series of diluted FBS in PBS (37 °C, 30 min). Data were quantified from gel electrophoresis by normalizing the band densities of FBS-treated circRNA to that of PBS-treated circRNA (t-test). RNA (100 nM) was transfected using Lipofectamine 3000 unless denoted otherwise. Data represent mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Bonferroni post-test.

Fig. 3 ∣. LNPs efficiently delivered small circRNA to lymph nodes and APCs to elicit T cell responses.

a, Design of nanocarrier screening for small circRNA vaccines, using circRNA-SIINFEKL as a model and 5moU-mRNA-OVA as a control. RNA: 5 μg, subcutaneous (s.c.) injection at the tail base of C57BL/6 mice (n = 5) on days 0 and 14. b, Tetramer staining on day 21 showed that SM-102 LNPs of circRNA-SIINFEKL elicited the highest frequency of PBMC SIINFEKL⁺CD8⁺ T cells among all these RNA nanoformulations. c, Luminex results of normalized serum cytokine and chemokine levels 12 h after booster immunization (day 14). SM-102 LNP-circRNA-SIINFEKL induced relatively low reactogenicity-associated chemokines. Each cytokine or chemokine level ($X$) was respectively normalized as follows: $X_{\text{normalized}}=\frac{X-X_{\text{min}}}{X_{\max }-X_{\text{min}}}$. d–f, Upon s.c. injection at the foot pad of BALB/c mice (n = 5), SM-102 LNPs efficiently delivered IR800-circRNA-SIINFEKL (0.5 nmol) to draining popliteal lymph nodes (dLNs) (circled in d), as shown by whole-body IVIS imaging (d), quantified IR800 fluorescence intensities of popliteal dLNs (e) and tissue fluorescence intensities quantified from ex vivo IVIS imaging (f). g, Flow cytometry results showing the Cy5-circRNA⁺ APC subsets among total CD45⁺ cells in draining lymph nodes 24 h after s.c. injection of free Cy5-circRNA or Cy5-circRNA LNPs, respectively. h, Confocal microscopy images showing efficient LNP delivery of Cy5-circRNA to DC2.4 cells and rapid endosome escape of Cy5-circRNA, the latter of which was indicated by the cytosolic Cy5-circRNA outside endolysosomes (circRNA, 100 nM; treatment, 0.5 h). Inset: one cell. i, As quantified from the above confocal microscopy images, the Cy5-circRNA fluorescence signal intensity ratios of outside/inside (O/I) endolysosome suggest the rapid endosome escape of LNP-circRNA in DC2.4 cells. Liposomal circRNA (lipo-circRNA) served as a control.

Fig. 4 ∣. Small circRNA vaccine activated PRRs for intrinsic immunostimulation with low PKR activation.

a–c, Gene transcriptome analysis results from BMDCs LNP-transfected with circRNA-SIINFEKL, 5moU-mRNA-OVA and PBS, respectively (24 h). The log₂-transformed fold change (FC) represents log₂(ratio of the mean expression induced by vaccine relative to PBS) (n = 3). a, Transcription heatmaps of genes involved in inflammation, migration, antigen processing and presentation, TLRs, RLRs, CLRs and miscellaneous immune-related genes. b, Volcano plot of differentially accessible peaks between blank LNPs and circRNA-SIINFEKL LNPs. c, log₂(FC) of specific genes of interest related to the indicated pathways. d, PRR reporter cell activities upon treatment with circRNA-SIINFEKL (100 nM, 24 h) and controls. Poly(I:C) served as a positive control. e, RT–PCR results of Ifnb levels in wild-type (WT) RM1 and RM1-IPS1-KO cells treated with circRNA-SIINFEKL (100 nM, 48 h). f, Western blot analysis of PKR and pPKR in HEK293T cells after transfection with PBS, small circRNA-SIINFEKL, large circRNA-RBD and poly(I:C) positive control (0.5 μg per well), respectively, for 4 h. g, Western blot intensity ratio of pPKR to β-actin in treated HEK293T cells. Relative phosphorylation is indicated, calculated as the band intensity ratio ($X$) of pPKR to β-actin and then normalized to the relative phosphorylation induced by no RNA treatment group. $X_{\text{normalized}}=\frac{\left({\text{Intensity}}_{\text{pPKR}}∕{\text{Intensity}}_{\beta -\text{actin}}\right)\text{for RNA}}{\left({\text{Intensity}}_{\text{pPKR}}∕{\text{Intensity}}_{\beta -\text{actin}}\right)\text{for PBS}}$. h, HEK293T cell viability transfected with the indicated RNA (0.5 μg per well, 24 h) or PBS control. Cell viability was normalized to PBS-treated cells. In d–h, RNA was transfected using Lipofectamine 3000. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA with Bonferroni post-test. NSm not significant.

Fig. 5 ∣. Small circRNA vaccine outperformed several benchmark modified mRNA and large circRNA vaccines to elicit robust and durable T cell responses with great safety in young adult mice and aged mice.

a–h, Benchmark studies of small circRNA vaccine versus three types of modified mRNA vaccine, an unmodified mRNA vaccine (no nucleoside modification), and a large circRNA vaccine in DCs and young adult mice. a, Three-day AUC of the H-2K^b-SIINFEKL MFI on DCs LNP-transfected with circRNA-SIINFEKL and three modified mRNAs, respectively. Data were quantified from flow cytometry results. Asterisks: statistical significance relative to circRNA. b, Timeline of in vivo benchmark study. C57BL/6 mice (6–8 weeks) were immunized with circRNA-SIINFEKL, as well as three modified mRNAs, unmodified mRNA-OVA and large circRNA-OVA, at escalating doses, respectively. c–e, Benchmarking circRNA-SIINFEKL against three types of modified mRNA for their ability to elicit T cell responses in mice (n = 9 for PBS, circRNA-SIINFEKL and 5moU-mRNA-OVA; n = 4–5 for the other groups). c, Tetramer staining results on day 21 suggest that circRNA-SIINFEKL elicited higher fractions of PBMC SIINFEKL-specific T cells than all modified mRNAs. circRNA-SIINFEKL elicited dose-dependent T cell responses, which plateaued at ca. 60% (dose 2 × 30 μg). Asterisks: statistical significance relative to circRNA. d, circRNA-SIINFEKL caused less mouse body weight drop and more rapid weight recovery than modified mRNAs, as exemplified 1–2 days after the 2nd 30 μg dose. e, 180-day kinetics of the PBMC SIINFEKL-specific CD8⁺ T cell percentages in the immunized mice, suggesting that circRNA-SIINFEKL elicited more potent and more durable T cell responses than these modified mRNAs, at all corresponding doses before the T cell responses plateaued. Asterisks denote statistical significance of the T cell fraction AUC relative to that for circRNA. f, Estimated total counts of peripheral and splenic SIINFEKL-specific CD8⁺ T cells per mouse (day 35, day 180). circRNA-SIINFEKL (3 × 10 μg) elicited SIINFEKL-specific CD8⁺ T cell counts that are >2-fold typical doses of adoptive TCR-T cells (5 × 10⁶, dashed line) used for mouse tumour immunotherapy. g, Tetramer staining results on day 21 and day 35 suggest that circRNA-SIINFEKL elicited higher fractions of PBMC SIINFEKL-specific T cells than unmodified mRNA-OVA and large circRNA-OVA. Asterisks: statistical significance relative to circRNA. C57BL/6 mice (6–8 weeks) were subcutaneously immunized with circRNA-SIINFEKL (n = 9), unmodified mRNA-OVA (n = 4) and large circRNA-OVA (n = 4) (dose 3 μg and 10 μg, days 0, 14 and 28). h, Luminex heatmaps showing the serum chemokine and cytokine levels 6 h after the third immunization (day 28). Each cytokine or chemokine level ($X$) was independently normalized for each dose as follows: $X_{\text{normalized}}=\frac{X-X_{\text{min}}}{X_{\max }-X_{\text{min}}}$. Overall, circRNA-SIINFEKL induced the least reactogenicity-associated chemokine and cytokine levels among all vaccines. i–k, Low-dose circRNA-SIINFEKL (5 μg; days 0, 14 and 28) elicited potent and durable T cell immunity in immunosenescent aged mice (1 year old; n = 5). i, Tetramer staining showed superior PBMC SIINFEKL-specific CD8⁺ T cell responses elicited by circRNA-SIINFEKL than 5moU-mRNA-OVA or CpG-adjuvanted OVA (day 21) (t-test). j, Intracellular IFNγ and TNF staining results in PBMC CD8⁺ T cells from the above immunized aged mice (day 35). T cells were restimulated with SIINFEKL peptide. k, circRNA-SIINFEKL vaccine protected immunized aged mice from EG7-OVA tumour cell challenge (1 × 10⁶ cells, s.c. administration on day 70) (asterisks denote statistical significance relative to circRNA). Vaccines were delivered by SM-102 LNPs and subcutaneously injected at mouse tail base. Data represent mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA with Bonferroni post-test, unless denoted otherwise.

Fig. 6 ∣. Broad application of small circRNA vaccines to elicit T cell responses against various peptide antigens.

a, Design of T cell response study for small circRNAs encoding various types of peptide antigen in C57BL/6 mice (6–8 weeks; n = 5). RNA, 5 μg per RNA; CpG, 5 μg; peptides, 10 μg; s.c. injection at tail base. Vaccines were delivered by LNPs. T cells were restimulated with OVA for intracellular cytokine staining. b, Intracellular IFNγ and TNF staining showed that MHC-II-restricted circRNA-ISQ elicited effector CD4⁺ T cells in PBMCs (day 21). 5moU-mRNA-OVA served as a benchmark. c, Intracellular IFNγ and TNF staining showed that MHC-II-restricted circRNA-ISQ enhanced the ability of MHC-I-restricted circRNA-SIINFEKL to elicit SIINFEKL-specific CD8⁺ T cell response (day 21). Both monovalent and bivalent circRNAs outperformed 5moU-mRNA-OVA to elicit the corresponding antigen-specific CD4⁺ or CD8⁺ T cell responses. d–k, Small circRNA vaccines elicited T cell responses against various cancer and viral peptide antigens. d, Tetramer staining results showed the fractions of ADPGK ⁺CD8⁺ T cells among all live PBMC CD8⁺ T cells, indicating that circRNA-ADPGK neoantigen vaccine elicited robust T cell responses. e, circRNA-ADPGK elicited T cell memory (day 70). f, circRNA-ADPGK elevated PD-1 expression on CD8⁺ T cells (day 21). g, Intracellular IFNγ and TNF staining of PBMC CD8⁺ T cells (day 21) from C57BL/6 mice immunized with circRNA-TRP2/gp100 or CpG-adjuvanted peptide vaccines (days 0 and 14). h, The frequencies of PBMC E7⁺CD8⁺ T cells over 70 days post priming from C57BL/6 mice immunized with circRNA-E7_43–62 or CpG-adjuvanted peptide vaccines (days 0, 14 and 28). i, Intracellular IFNγ and TNF staining of PBMC CD8⁺ T cells (day 21) from the above immunized mice. j, Tetramer staining results showing the fractions of RBD_440–459⁺CD8⁺ T cells among all live PBMC CD8⁺ T cells from as-immunized C57BL/6 mice (days 0, 14 and 28), indicating that circRNA-RBD_440–459 elicited potent RBD_440–459-specific CD8⁺ T cell responses. k, Intracellular IFNγ and TNF staining of PBMC CD8⁺ T cells (day 21) from the above immunized mice (asterisks in d, h and j: statistical significance relative to circRNA). Vaccines: subcutaneously injected at mouse tail base. Data represent mean ± s.e.m. (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA with Bonferroni post-test unless denoted otherwise.

Fig. 7 ∣. Small circRNA neoantigen vaccine reduced tumour immunosuppression for potent tumour immunotherapy.

a, Study design of TME immune analysis and tumour immunotherapy in mice. MC38 tumour cells were subcutaneously inoculated in the flank of C57BL/6 mice, and treatment started when tumours reached around 60 mm³ on day 6. b–d, MC38 tumour immune microenvironment analysis (day 15) upon treatment with circRNA-ADPGK, alone or combined with anti-PD-1 (n = 6–8). 5moU-mRNA-ADPGK served as a control. b, The percentage of different immune cells among CD45⁺ cells in TME after the indicated treatment. c, Tetramer staining results showed the fractions of ADPGK⁺CD8⁺ T cells among total live CD8⁺ T cells in TME, indicating that circRNA-ADPGK vaccine combined with anti-PD-1 enhanced antigen-specific cytotoxic T cells within the TME. d, The ratio of CD8⁺ T cells to T_reg cells within the TME. e–g, RNA-seq transcriptome analysis of MC38 tumours (day 15) after the above treatment (n = 3). e, Transcription heatmaps of selected genes related to immune modulation. f, The log-transformed mean expression ratio (log₂(FC) of pathway-related genes in immunotherapy-treated tumours compared with that in PBS-treated tumours. g, Triwise radar plots depicting Gene Ontology enrichment analysis of T cell priming and DC activation pathways (left) and regulation of T cell proliferation (right). Black dots, all genes; red dots, genes related to the corresponding immune functions. h, Low-dose circRNA-ADPGK (5 μg) for potent combination immunotherapy of MC38 tumour, as shown by MC38 tumour growth and Kaplan–Meier mouse survival curves. liRNA and 5moU-mRNA encoding the same peptide antigen were used as controls (n = 14 for PBS, anti-PD-1, circRNA-ADPGK and circRNA-ADPGK + anti-PD-1; n = 7 for the other groups). Asterisks denote statistical significance between the AUC of circRNA-ADPGK tumour growth and that of circRNA-ADPGK + anti-PD-1. P = 0.0480: statistical analysis between the AUC of circRNA-ADPGK tumour growth and that of mRNA-ADPGK. i, MC38 tumour volumes after lymphocyte depletion using anti-CD8, anti-CD4 or anti-NK1.1 antibodies. RNA vaccines were delivered by SM-102 LNPs and subcutaneously injected at tail base. RNA, 5 μg; antibodies, 100 μg, intraperitoneal (i.p.) injection. Data represent mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA with Bonferroni post-test unless denoted otherwise.

Fig. 8 ∣. Monovalent or multivalent small circRNA vaccines for robust combination immunotherapy of multiple types of tumour.

a, Design of tumour immunotherapy studies in mice. Tumour cells were inoculated subcutaneously in mouse flank, and treatment started when tumour volumes were around 50 mm³. Vaccines were loaded in LNPs and subcutaneously injected at tail base; antibodies were intraperitoneally injected. b, Average TC-1 tumour volumes after treatment with circRNA-E7_43–62 + anti-PD-1 and controls (n = 6–7). c, Spider plots of individual TC-1 tumour growth curves and complete regression (CR) rates after the above treatment. In b and c, vaccine, 5 μg RNA, 5 μg CpG, 10 μg E7_43–62 peptide antigens; anti-PD-1, 200 μg. Asterisks: statistical significance relative to circRNA + ICB. d, Average volumes of B16F10 melanoma treated with MHC-I/II-restricted tetravalent circRNA-T2/g/T1/T1 vaccine, alone or combined with anti-PD-1 + anti-CTLA-4. Asterisks denote statistical significance relative to circRNA-T2/g/T1/T1. e, B16F10 melanoma tumour growth after circRNA-T2/g/T1/T1 vaccine treatment and lymphocyte depletion. Asterisks: statistical significance relative to circRNA-T2/g/T1/T1. f, Spider plots of individual B16F10 melanoma tumour growth curves and CR rates. g, Kaplan–Meier survival curves of the as-treated B16F10 melanoma-bearing mice. Asterisks: statistical significance relative to circRNA + ICB. In d–g, circRNA, 10 μg; antibodies, 100 μg per ICB antibody, 200 μg anti-CD8, anti-CD4 or anti-NK1.1. h, Average (left) and individual (right) tumour growth curves of Braf^V600E SM1 melanoma treated with circRNA-T2/g/T1/T1 combined with anti-PD-1 + anti-CTLA-4, as well as controls. Asterisks: statistical significance relative to circRNA-T2/g/T1/T1. i, Kaplan–Meier survival curves of Braf^V600E SM1 melanoma-bearing mice treated as above. Asterisks: statistical significance relative to circRNA + ICB. In h and i, circRNA, 30 μg; ICB antibodies, 200 μg each. Vaccines: loaded in SM-102 LNPs and subcutaneously injected at mouse tail base. Data represent mean ± s.e.m. (n = 6–8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA with Bonferroni post-test unless denoted otherwise.