Abstract
Intracellular DNA sensors regulate innate immunity and can provide a bridge to adaptive immunogenicity. However, the activation of the sensors in antigen-presenting cells (APCs) by natural agonists such as double-stranded DNAs or cyclic nucleotides is impeded by poor intracellular delivery, serum stability, enzymatic degradation and rapid systemic clearance. Here we show that the hydrophobicity, electrostatic charge and secondary conformation of helical polypeptides can be optimized to stimulate innate immune pathways via endoplasmic reticulum stress in APCs. One of the three polypeptides that we engineered activated two major intracellular DNA-sensing pathways (cGAS–STING (for cyclic guanosine monophosphate–adenosine monophosphate synthase–stimulator of interferon genes) and Toll-like receptor 9) preferentially in APCs by promoting the release of mitochondrial DNA, which led to the efficient priming of effector T cells. In syngeneic mouse models of locally advanced and metastatic breast cancers, the polypeptides led to potent DNA-sensor-mediated antitumour responses when intravenously given as monotherapy or with immune checkpoint inhibitors. The activation of multiple innate immune pathways via engineered cationic polypeptides may offer therapeutic advantages in the generation of antitumour immune responses.
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Data availability
The data supporting the findings of this study are available within the article 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 are provided with this paper.
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Acknowledgements
We thank C. Wogan of the Division of Radiation Oncology, MD Anderson Cancer Center, for editorial assistance. This work is supported in part by the National Cancer Institute grant (1K08 CA241070) to W.J., US Department of Defense grant (W81XWH-19-1-0325) to B.Y.S.K. and the Cancer Center Support (Core) grant (P30 CA016672) from the National Cancer Institute, National Institutes of Health, to The University of Texas MD Anderson Cancer Center (principal investigator P.W. Pisters).
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D.Y.L., B.Y.S.K. and W.J. conceived the project and designed experiments. B.Y.S.K. and W.J. supervised the project. D.Y.L. carried out all the experiments and analysed all the data. Y.W., K.H., Y.L., M.K., S.D.J. and Y.Q. performed the in vivo studies. T.M.L. and J.A.T. performed size exclusion chromatography for molecular weight determination. Y.W., X.L., S.D., Y.A. and J.L. helped to interpret the data. D.Y.L., W.J. and B.Y.S.K. wrote the paper with help from all authors.
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Extended data
Extended Data Fig. 1 Varying hydrophobicity in the engineered polypeptide affects immunogenicity via ER stress-mediated mtDNA release and effector functions in macrophages.
a, Chemical structure of cationic polypeptides with different amine-containing analogues. Cationic polypeptides including hydrophilic analogues and cyclic structures more favorably induced. (b) ER stress and (c) mtDNA release in BMDMs (n = 3). The representative western blot images were shown from at least twice independent results. Cationic polypeptide tethered with a hydrophilic building block and cyclic structure increased (d) phagocytosis of EO771 breast cancer cells and (e) cross-presentation of the model antigen SIINFEKL-H2Kb (n = 3). (f) Gene expression of pro-inflammatory cytokines was affected by hydrophobicity of polypeptides and the chemical structure of amine-including analogues (n = 3). One-way ANOVAs with Bonferroni post hoc correction were used in c, d, e, f. All data are expressed as means±s.d.
Extended Data Fig. 2 Varying the electrostatic charge of the polypeptide affects immunogenicity via ER stress-mediated mtDNA release and effector functions in macrophages.
a, Chemical structure of polypeptides with different electrolytes. P1 more favorably induced (b) ER stress and (c) mtDNA release in BMDMs greater than did PTMA (strongly cationic) and PS (anionic) (n = 3). The representative western blot images were shown from at least twice independent results. P1 improved (d) phagocytosis of EO771 breast cancer cells and (e) cross-presentation of the model antigen SIINFEKL-H2Kb to a greater extent than did PTMA or PS (n = 3). f, Expression of genes for pro-inflammatory cytokines was regulated by types of electrolytes and strength of cationic charges (n = 3). One-way ANOVAs with Bonferroni post hoc correction were used in c, d, e, f. All data are expressed as means±s.d.
Extended Data Fig. 3 Varying the length of the side chain of the polypeptide regulates immunogenicity via ER stress-mediated mtDNA release and effector functions in macrophages.
a, Chemical structures of polypeptides with side chains of different lengths. P1 induced (b) ER stress and (c) mtDNA release in BMDMs more strongly than did P3 (mid-length) and PS (short length) (n = 3). The representative western blot images were shown from at least twice independent results. P1 improved (d) phagocytosis of EO771 breast cancer cells and (e) cross-presentation of the model antigen SIINFEKL-H2Kb to a greater extent than did P3 and P1 (n = 3), ordinary one-way ANOVA. f, Expression of genes for pro-inflammatory cytokines was affected by the length of side chains in the polypeptides (n = 3). One-way ANOVAs with Bonferroni post hoc correction were used in c, d, e, f. All data are expressed as means±s.d.
Extended Data Fig. 4 P1 promotes innate and adaptive immune activation within the tumour microenvironment.
a, P1 increased the population of tumour-infiltrating CD8+ T cells and IFNγ-producing CD8+ T cells, but decreased that of Treg cells (CD3+CD4+CD25+FoxP3+), relative to P2 and P3 (n = 3 biological replicates). Tumour-infiltrating lymphocytes were obtained on day 16 (2 days after the last treatment). b, P1 promoted M1 macrophage polarization (M1 macrophage: CD80+CD86+CD206– ; M2 macrophage: CD80–CD206+; macrophages: CD11b+CD11c–F4/80+) and maturation of dendritic cells (DCs) (Mature DCs: CD80+CD86+ DC; DC: CD11c+MHC-II+F4/80–) but did not affect the number of MDSCs (CD11b+CD11c–MHC-II–F4/80–Gr-1+) within the tumour microenvironment relative to P2 and P3 (n = 3), unpaired two-tailed Student’s t test in comparison with Cont and the indicated conditions. All data are expressed as means±s.d.
Extended Data Fig. 5 P1 combined with αPD1 evoked strong antitumour immunity in mice bearing metastatic tumours.
a, Timeline for tumour establishment and administration of P1 and αPD1. b, Growth curves for 4T1 tumours in mice after the indicated treatments (n = 8 biologically independent mice for HEPES; n = 9 biologically independent mice for P1; n = 10 biologically independent mice for P1 + αPD1 and cGAMP+αPD1), unpaired two-tailed Student’s t test in comparison with HEPES or the indicated conditions on day 30 after tumour inoculation. c, Photographs of excised 4T1 tumour tissues on day 17. d, Kaplan-Meier survival curves of 4T1 tumour-bearing mice; log-rank (Mantel-Cox) test in comparison with HEPES or the indicated conditions. e, Flow cytometry of tumour-infiltrating T lymphocytes (Treg cells: CD3+CD4+CD25+FoxP3+) and their subsets (n = 4 biological replicates), which were harvested on day 17. f, Immunofluorescence stains for CD4, CD8, and Iba1 in macrophages. Representative images from random fields of view in one of the three biologically independent mice. Scale bar, 30 μm. g. Profiles of tumour-infiltrating myeloid cells (M1 macrophage: CD80+CD86+CD206−; M2 macrophage: CD80−CD206+ macrophage: CD11b+CD11c−F4/80+; mature DC: CD80+CD86+ DC; DC: CD11c+MHC-II+F4/80−; MDSC: CD11b+CD11c−MHC-II−F4/80−Gr-1+) as evaluated by flow cytometry (n = 4 biological replicates); unpaired two-tailed Student’s t test in comparison with Cont or the indicated condition. All data are expressed as means±s.d.
Extended Data Fig. 6 P1 combined with αPD1 generates robust systemic antitumour immunity.
a, P1 + αPD1 treatment increased the production of IFNγ from CD8+ T cells and decreased the population of Treg cells (CD3+CD4+CD25+FoxP3+) in tumour-draining lymph nodes and spleen as compared with the other treatment conditions (n = 4 biological replicates), unpaired two-tailed Student’s t test in comparison with HEPES or the indicated conditions. b, P1 + αPD1 treatment increased the population of mature DCs (CD11c+F4/80−MHC-II+CD80+CD86+) and pro-inflammatory macrophages polarization (CD80+CD86+CD11b+CD11c−F4/80+ for migratory macrophages in spleen, CD80+CD86+CD11b+CD11c–F4/80+ for macrophages in lymph nodes), but maintained the numbers of MDSCs (CD11b+CD11c-F4/80-MHC-II−Gr-1+) in tumour-draining lymph nodes and spleen, relative to the other treatments (n = 4 biological replicates); unpaired two-tailed Student’s t test in comparison with HEPES or the indicated conditions. All data are expressed as means±s.d.
Extended Data Fig. 7 Activation of MyD88 and STING is required to stimulate the p-IRF3 axis in tumour-homing DCs treated with P1 + αPD1.
Immunofluorescence images show that DCs treated with P1 + αPD1 promoted p-IRF3 nuclear translocation (white arrows) in WT but not in STING–/– or MyD88–/– cells. Scale bar, 30 μm. One-way ANOVA with Bonferroni post hoc correction was used to determine statistical significance. Numbers of nucleus-translocating p-IRF3+ DCs in the immunofluorescence images were counted with ImageJ software (n = 6 biological replicates). All data are expressed as means±s.d.
Extended Data Fig. 8 TLR9 activation is required to recruit tumour-infiltrating T cells by stimulating IRF7 signaling in tumour-homing APCs treated with P1 + αPD1.
a, P1 + αPD1 treatment promoted p-IRF7 nuclear translocation in macrophages and DCs in WT but not in TLR9–/– mice. Scale bar, 30 μm; unpaired two-tailed Student’s t test in comparison to the indicated conditions (n = 8 biological replicates). b, P1 + αPD1 treatment of TLR9–/– mice blocked recruitment of both CD4+ and CD8+ T cells into tumours. Scale bar, 20 μm; unpaired two-tailed Student’s t test in comparison to the indicated conditions (n = 6 biological replicates). Numbers of cells in the immunofluorescence images were counted by ImageJ software. All data are expressed as means±s.d.
Extended Data Fig. 9 Activation of STING and MyD88 in cancer cells is not required for P1 + αPD1’s antitumour effect.
a, Immunoblots verifying that EO771 STING–/– cells did not express STING, and EO771 MyD88–/– cells did not express MyD88. The representative western blot images are shown from at least two independent experiments. b, Tumour growth curves for mice with EO771 WT, EO771 STING–/–, or EO771 MyD88–/– tumours after intravenous injection of HEPES or P1 + αPD1 (n = 5 biologically independent mice for all treatment groups). c, Kaplan-Meier survival curves for mice with EO771 WT, EO771 STING–/–, or EO771 MyD88–/– tumours; log-rank (Mantel-Cox) test compared with the indicated conditions. All data are expressed as means±s.d.
Extended Data Fig. 10 CD8+ T-cell depletion abolished adaptive immunity conferred by P1 + αPD1.
a, CD8+ T cell populations in splenocytes, evaluated by flow cytometry on day 17 after tumour cell inoculation (n = 3 biological replicates). (b, c) CD8 depletion (b) abrogated the antitumour effect of P1 + αPD1 and (c) reduced the survival of EO771 tumour-bearing mice (n = 6 biologically independent mice for all treatment groups); unpaired two-tailed Student’s t test in comparison to P1 + αPD1 at day 24 after tumour inoculation for tumour growth curve; log-rank (Mantel-Cox) test for Kaplan-Meier survival curve. d, Excised tumours from each experimental group show that CD8 depletion eliminated the antitumour effect of P1 + αPD1. e, Immunofluorescence stains of tumour-infiltrating CD8+ T cells show that CD8 depletion inhibited the recruitment of CD8+ T cells within tumours. Scale bar, 30 μm. f, CD8 depletion reduced the proportions of tumour-infiltrating CD8+ T cells and IFNγ+CD8+ T cells, but did not change the proportion of Tregs (n = 3 biological replicates); unpaired two-tailed Student’s t test; n.s., not significant compared with the indicated conditions. All data are expressed as means±s.d.
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Supplementary Data 1
Statistical data for Supplementary Figs. 4, 5, 7–19, 22–25 and 27–36.
Supplementary Data 2
Uncropped western blots for Supplementary Figs. 12, 22 and 24–29.
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Source Data Figs. 1–8 and Extended Data Figs. 1–10
Statistical source data for Figs. 1–8 and Extended Data Figs. 1–10.
Source Data Fig. 3 and Extended Data Figs. 1–3 and 9
Uncropped western blots for Fig. 3 and Extended Data Figs. 1–3 and 9.
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Lee, D., Huntoon, K., Wang, Y. et al. Synthetic cationic helical polypeptides for the stimulation of antitumour innate immune pathways in antigen-presenting cells. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-024-01194-7
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DOI: https://doi.org/10.1038/s41551-024-01194-7
