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Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate established tumors and prime systemic immunity

Abstract

Therapies that synergistically stimulate immunogenic cancer cell death (ICD), inflammation and immune priming are of great interest for cancer immunotherapy. However, even multi-agent therapies often fail to trigger all of the steps necessary for self-sustaining antitumor immunity. Here we describe self-replicating RNAs encapsulated in lipid nanoparticles (LNP), which combine three key elements: (1) an LNP composition that potently promotes ICD, (2) RNA that stimulates danger sensors in transfected cells and (3) RNA-encoded interleukin (IL)-12 for modulation of immune cells. Intratumoral administration of LNP-replicons led to high-level expression of IL-12, stimulation of a type I interferon response and cancer cell ICD, resulting in a highly inflamed tumor microenvironment and priming of systemic antitumor immunity. In several mouse models of cancer, a single intratumoral injection of LNP-replicons eradicated large established tumors, induced protective immune memory and enabled regression of distal uninjected tumors. LNP-replicons are thus a promising multifunctional single-agent immunotherapeutic.

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Fig. 1: LNP-replicon formulations induce high levels of payload gene expression and immunogenic cell death.
Fig. 2: TT3 LNPs carrying replicon RNA induce immunogenic cell death and immune infiltration of tumors in vivo.
Fig. 3: LNP-replicons encoding IL-12-alb or IL-12-alb-lum remodel the TME.
Fig. 4: A single injection of LNP-replicons encoding IL-12-alb or IL-12-alb-lum can eradicate large established tumors.
Fig. 5: Local LNP-replicon therapy regresses distal untreated tumors and eliminates metastases.
Fig. 6: Cellular and molecular pathways governing LNP-replicon therapeutic efficacy.

Data availability

The source data for Figs. 16 and Extended Data Figs. 12 and Figs. 47 have been provided as source data files (except for Fig. 3g, which are stored at https://doi.org/10.6084/m9.figshare.12502007). The source data for Fig. 5h are also stored at https://doi.org/10.6084/m9.figshare.12502124. The source data for Extended Data Fig. 1d–g are also stored at https://doi.org/10.6084/m9.figshare.12498047. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank the Koch Institute Swanson Biotechnology Center for technical support, especially the Flow Cytometry Facility, Animal Facility, Microscopy Facility, Histology Facility and Nanotechnology Materials Core Facility. We also thank G.A. Paradis, J. Kuhn, D.S. Yun and members of Irvine laboratory and members of Weiss laboratory for their discussions and suggestions. This work was supported in part by the grant P30-CA14051 from National Cancer Institute in support of Koch Institute core facilities, the National Institutes of Health (award CA20618 to R.W. and D.J.I.), the Marble Center for Nanomedicine and the Ragon Institute of MGH, MIT and Harvard. D.J.I. is an investigator of the Howard Hughes Medical Institute. Y.D. acknowledges support from the Maximizing Investigators’ Research Award R35GM119679 from the National Institute of General Medical Sciences. W.Z. acknowledges the support from the Professor Sylvan G. Frank Graduate Fellowship.

Author information

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Authors

Contributions

Y.L., D.J.I. and R.W. designed the study and wrote the manuscript. Y.L., Z.S., W.Z. and X.Z. performed the experiments. N.M. and K.D.W. provided IL-12-alb and IL-12-alb-lum constructs and IL-12-alb and IL-12-alb-lum proteins, and helpful discussions. W.Z., C.Z. and Y.D. synthesized TT3 and provided technical support for the TT3 LNP formulations and analyzed the data.

Corresponding authors

Correspondence to Yizhou Dong or Darrell J. Irvine or Ron Weiss.

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Competing interests

D.J.I., K.D.W., R.W., Y.L., N.M. and Y.D. are named as inventors on patent applications filed by MIT related to the data presented in this work (PCT/US2020/013069).

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Extended data

Extended Data Fig. 1 Comparison of size, zeta potential, RNA encapsulation efficiency, and morphology for the nanoparticles.

a, b, Mean diameter (a) (nm) and Zeta potential (b) (mv) of different nanoparticles with or without encapsulation of replicon RNA. Shown are mean (box) of three different measurements from one of two independent experiments. (c) RNA encapsulation efficiency of DOTAP, Lipofectamine, and TT3 nanoparticles. Shown are mean (box) of 3 different measurements from one of two independent experiments. n.d. means non-detectable. dg, morphologies of DOTAP and TT3 lipid nanoparticles without (d, f) or with replicon RNA (e, g) were imaged by Cryo-TEM. Shown are representative images from one of three independent fields of view in a single experiment. Scale bar (100 nm) is indicated. The source data are provided with this paper; the source data for dg are also stored at https://doi.org/10.6084/m9.figshare.12498047.

Source data

Extended Data Fig. 2 Immunogenic cell death in human cancer cells and pathways of interferon stimulation in response to treatments with TT3 or TT3-Rep.

ac, Human A549 lung carcinoma (a), SK-MEL-5 melanoma (b), or Hela cervical cancer cells (c) were treated with 10 µg/mL empty TT3 LNPs (TT3), TT3 LNPs carrying replicons encoding GFP (TT3-Rep), or left untreated as indicated. Three days post treatment, the percentage of viable cells was enumerated (left column). One day post treatment, the ICD markers extracellular ATP (second column), CRT+ cells (third column), extracellular HMGB1 (fourth column) were measured. Shown are mean (box) of different treatments from 3 technical cell culture replicates from a single experiment. d, Twelve hours post treatment of 10 µg/mL TT3 LNP (LNP) or TT3 LNP carrying replicons encoding mCherry (LNP-Rep), levels of MDA-5 and TLR3 mRNA in B16F10 cells were assessed by qPCR. Shown are fold changes of MDA-5 and TLR3 expression in different treatments (n = 6 from 3 technical cell culture replicates and 2 technical qPCR replicates) in comparison to untreated cells (n = 6 from 3 technical cell culture replicates and 2 technical qPCR replicates) normalized by actin B expression from a single experiment. e, One day post treatment of A549 type I interferon reporter cells or MDA-5 knockout A549 reporter cells with 10 µg/mL TT3 LNP (LNP) or TT3 LNP carrying replicons encoding mCherry (LNP-mCherry), the supernatants were collected and incubated to detect stimulation the expression of the luciferase reporter. Shown are relative luminescence units from n = 4 technical cell culture replicates from a single experiment.

Source data

Extended Data Fig. 3 Flow cytometry gating strategy for lymphocyte analysis.

C57Bl/6 mice bearing established B16F10 tumors and TDLNs were necropsied and prepared as single cells suspension. The cells were stained with antibodies against cell surface makers as indicated and followed with flow cytometry (see methods). a. Shown are gating strategies of granulocytes, M-MDSCs, monocytes, macrophages, CD4 T cells, CD8 T cells, NK cells, NKT cells, mCherry positive cells in tumor cells for Fig. 2d–g and Fig. 3e. b. Shown are typical gating strategies of CRT positive cells in SSChi FSChi live tumor cells from B16F10 tumor bearing mice treated without or with LNP-Rep for Fig. 2h.

Extended Data Fig. 4 Treatments of LNP-Rep encoding mCherry, IL-12-alb, or IL-12-alb-lum reprogrammed tumor and systemic microenvironments with low toxicity.

a, IL12-alb-lum produced by LNP-replicon-transfected tumor cells binds to collagen I. B16F10 cells were transfected with LNP-Rep(IL-12-alb-lum) and the supernatants of the transected cells were added to collagen-coated plates for analysis of binding by ELISA in comparison of standards protein IL12-alb-lum. Shown are the ELISA absorbance for IL-12 detection versus concentration of added IL-12-alb-lum using the supernatants of B16F10 cells that were transfected with LNP-Rep(IL-12-alb-lum) or left untreated (n = 4 technical cell culture replicates from one of two independent experiments. b, c, Comparison of AST (b) and ALT (c) levels in serum at day 1 and 3 after the indicated treatments (error bars are mean + s.e.m. from n = 5 mice/group). d, Comparison of CXCL9 and CXCL10 levels in tumors at day 1 and 3 after the indicated treatments (error bars are mean + s.e.m. from n = 5 mice/group). P values were determined by two way ANOVA analysis using PRISM Software and exact P values were indicated.

Source data

Extended Data Fig. 5 Therapeutic efficacy of LNP-Rep encoding IL-12 in distinct treatment models.

a,b, Groups of C57Bl/6 mice (n=10/group) bearing established B16F10 tumors were treated when tumors reached 50 mm2 with a single injection of 10 µg LNP-rep(IL-12-alb-lum) on day 7 (indicated by red arrows) or with 2.3 µg recombinant IL-12-alb-lum on days 5, 11, and 17 (indicated by blue arrows). Shown are average (mean + s.d.) tumor growth (a) and overall survival (b) over time. c,d, Groups of Balb/c mice bearing established CT26 tumors were treated when tumors reached 50 mm2 with a single injection on day 7 or 3 injections (day 7, 10, 13) of 10 µg LNP-rep(IL-12-alb-lum) starting from day 7 (untreated n=6 mice/group, other groups n=8 mice/group). Shown are average (mean + s.e.m.) tumor growth (c) and overall survival (d) over time. e–g, Groups of C57Bl/6 mice bearing established B16F10 tumors were treated when tumors reached 50 mm2 with a single injection of different dosages of the LNP-encapsulated replicon RNA as indicated. Shown are IL-12 levels (mean + s.e.m.) in tumors at one day post injection as measured by ELISA (n=5 mice/group (e), average (mean + s.d.) tumor growth (f) and overall survival (g) over time with the treatments as indicated (untreated n=6 mice/group, other groups n=8 mice/group). n.d., non-detectable. h–j, C57Bl/6 mice (untreated n=12 mice/group, other groups n=14 mice/group) were inoculated s.c. with 106 and 0.3x106 B16F10 cells in the left and right flanks, respectively. When the left flank tumor reached ~50 mm2 in size, this lesion was injected with 10 µg of the indicated LNP-replicon. Shown is tumor area (mean + s.d.) in each flank (h, i), and mice survival over time (j). P values were determined by one-sided Turkey’s multiple comparison test (Fig. 5f, h, i), or by one-sided Log-Rank (Mantel-Cox) test (Fig. 5b, d, g, j) using PRISM Software and exact P values were indicated.

Source data

Extended Data Fig. 6 Depletion efficiency in peripheral blood (a-e) and in tumors (f).

C57BL/6J mice bearing B16F10 tumors were administered the indicated depleting antibodies beginning one day prior to injection with 10 µg LNP-Rep(IL12-alb-lum). Then the depletion efficiency in peripheral blood (a-d, Untreated n = 4 mice, treated and untreated n = 5 mice/group; e, n=10 mice/group) and in tumor (f, n=5 mice/group) were determined by flow cytometer. Shown are typical FACS plots from the groups of Untreated, Treated (LNP-Rep(IL-12-alb-lum), and Treated with depleting antibodies as indicated. The mean and standard deviation of gated populations are indicated.

Source data

Extended Data Fig. 7 CD8 T cells and Batf3 are critical for therapeutic efficacy.

a, Mice treated as in Fig. 4b that rejected their primary tumor (Naïve n=12 mice/group, Cured (LNP-Rep(IL-12-alb)) n=14 mice/group, Cured (LNP-Rep(IL-12-alb-lum)) n=14 mice/group) were administered antibodies against CD8a beginning one day prior to rechallenge with 0.1x106 B16F10 cells. Then antibody against CD8a was administered every 3 days and followed for survival mice were observed. b, B16F10 tumor bearing mice in absence of Batf3 decreases responses to treatment of LNP-Rep(IL-12-alb). Shown are tumor areas over time after intratumoral injection of LNP-Rep encoding IL-12-alb into B16F10 tumors (mean + s.e.m.) in C57BL/6J or Batf3-/- mice (B6-Untreated n=5 mice/group, B6-LNP-Rep(IL-12-alb) n=7 mice/group, Baft3-/–Untreated n=5 mice/group, Baft3-/–LNP-Rep(IL-12-alb) n=6 mice/group). Throughout, P values were determined by one-sided Log-Rank (Mantel-Cox) test (a), or by one-sided Tukey’s multiple comparison test (b) using PRISM Software and exact P values were indicated.

Source data

Extended Data Fig. 8 Gating strategy for flow cytometry analysis of dendritic cells and pmel T cells.

C57Bl/6 mice bearing established B16F10 tumors and TDLNs were necropsied and prepared as single cells suspension. The cells were stained with antibodies against cell surface makers as indicated and followed with flow cytometry (see methods). a. Shown are gating strategies of conventional DC1 and DC2 cells in TDLNs for Fig. 6f. b. Shown are gating strategies of Pmel CD8 T cell activation by cells from inguinal lymph nodes for Fig. 6g.

Supplementary information

Supplementary Table 1.

Reporting Summary

Supplementary Table 2

Sequences of constructs for dead replicon RNA encoding EGFP (deRep-GFP)

Source data

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Source data for plots in Fig. 1.

Source Data Fig. 2

Source data and statistical analysis for plots in Fig. 2.

Source Data Fig. 3

Source data and statistical analysis for plots in Fig. 3a–f.

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Source data and statistical analysis for plots in Fig. 4.

Source Data Fig. 5

Source data and statistical analysis for plots in Fig. 5b–d,f–g.

Source Data Fig. 5

Raw images of Fig. 5h.

Source Data Fig. 6

Source data and statistical analysis for plots in Fig. 6.

Source Data Extended Data Fig. 1

Source data for plots in Extended Data Fig. 1a–c.

Source Data Extended Data Fig. 1

Raw images of Extended Data Fig. 1d–g.

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Source data and statistical analysis for plots in Extended Data Fig. 2.

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Source data and statistical analysis for plots in Extended Data Fig. 4.

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Source data and statistical analysis for plots in Extended Data Fig. 5.

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Source data and statistical analysis for plots in Extended Data Fig. 6.

Source Data Extended Data Fig. 7

Source data and statistical analysis for plots in Extended Data Fig. 7.

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Li, Y., Su, Z., Zhao, W. et al. Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate established tumors and prime systemic immunity. Nat Cancer 1, 882–893 (2020). https://doi.org/10.1038/s43018-020-0095-6

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