Research ArticleCancer

Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs

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Science Translational Medicine  30 Jan 2019:
Vol. 11, Issue 477, eaat9143
DOI: 10.1126/scitranslmed.aat9143
  • Fig. 1 In vivo OX40L expression detected in tumors and draining LNs after intratumoral mRNA administration.

    Expression of OX40L in MC38-R tumor–bearing mice after intratumoral injection with 5 μg of LNP-formulated mRNA. (A) Time course in tumor and liver lysates by enzyme-linked immunosorbent assay. Each data point is an individual sample (n = 3). (B) MC38-R cancer cells by flow cytometry by percentage or per cell by total MFI. (C and D) Myeloid cells within tumors (C) or TdLN (D). (E and F) Four DC types from tumors (E) or five in TdLN (F). Bars indicate mean with SD (B to F) [n = 8 except 3 for monocytes and 4 for macrophages in (D)]. *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001 by t test between control and OX40L mRNA treatment within each cell type (no significance where not shown) (B to F). Data are representative of three (A to D) or two (E and F) independent experiments. Control, control mRNA; h, hour; d, day.

  • Fig. 2 Induction of tumor regression in multiple tumor models by intratumoral mRNA treatment.

    (A) Profiles of baseline immune infiltrates in untreated H22, MC38-S, and MC38-R tumors (n = 6 to 8). (B) Tumor volumes of individual animals after OX40L or control mRNA treatment, 5 to 7.5 μg intratumorally per week (3× H22 and MC38-R, up to 6× MC38-S). (C and D) MC38-S (C) or MC38-R (D) tumors treated intratumorally with one (D, top) or four (C and D, bottom) mRNA doses per week (5 μg of individuals including control mRNA, 2.5 μg each of doublets, and 1.67 μg each of IL-23/IL-36γ/OX40L). Dashed vertical lines represent dosing days, and dashed horizontal lines represent study limits (B to D). CR, complete responder, out of group sample size. (E) Kaplan-Meier survival curves of MC38-S tumor–bearing animals of mRNA versus protein treatment. Statistical significance (*) between triplet mRNA and triplet protein by log-rank test. n = 10 (B, MC38-R; C), 12 (B, H22), 15 (B, MC38-S; D and E). Data are representative of three independent experiments (C), two independent experiments (A, B, and D), or one independent experiment (E).

  • Fig. 3 Inflammatory TME and early activation of the innate immune system induced by mRNA treatments.

    Response of MC38-R tumor–bearing mice to one 5-μg total dose of mRNA intratumorally (5 μg of individuals and 1.67 μg each of IL-23/IL-36γ/OX40L). (A and B) Time course of cytokines in tumors (A) and blood plasma (B) (n = 6). (C) CD86 on DCs in TdLN. Each point indicates one sample. (D) CD103+ DCs in TdLN, as total cells per LN. (E) DCs in tumors 7 days after treatment, per milligram of tumor tissue. n = 8 (C to E) [except 6 for control at 24 hours in (C) and (D) and 6 for control in (E)]. (F) Differential expression of DC transcripts by NanoString 7 days after treatment with IL-23/IL-36γ/OX40L versus control mRNA (n = 6). Statistical significance indicated within each time point between control mRNA and active mRNA (no significance where not shown): by analysis of variance (ANOVA) [^ in (C) and (D)], t test [* in (E)], or Benjamini-Yekutieli false discovery rate (FDR) [* in (F)]. (G) Survival of wild-type (WT) C57BL/6 or Batf3−/− MC38-S tumor–bearing animals treated with one mRNA dose (n = 10 for Batf3−/− or n = 15 for WT). Statistical significance (*) between treated WT and Batf3−/− by log-rank test. Data are representative of three independent experiments (E), two independent experiments (A to D and G), or one independent experiment (F).

  • Fig. 4 Induction of lymphocyte activation and remodeling of the TME by multi-mRNA treatment.

    Response of MC38-R tumor–bearing mice to one 5-μg total mRNA dose intratumorally (5 μg of individuals, 2.5 μg each of IL-23/IL-36γ, and 1.67 μg each of IL-23/IL-36γ/OX40L). (A and B) CD69 expression (A) or Ki-67 (B) on NKT and γδ T cells in TdLN. (C) Lymphocyte numbers in tumors. (D) Differential gene expression in tumors of lymphocyte cell type markers 7 days after IL-23/IL-36γ/OX40L treatment versus control mRNA. n = 6. (E and F) CD69 (E) or Ki-67 (F) on αβ CD4+ and CD8+ T cells in TdLN. n = 8 (A to C and E and F) [except 6 for control at 24 hours (A and E) and 5 for triplet at 7 days (C)]. (G) Lymphocyte numbers in tumors. EM, effector memory; CM, central memory; N, naïve. (H) Ratio of CD8+ T to Treg cells in tumors [n = 8 in (G) and (H), except 6 for control]. Statistical significance indicated (no significance where not shown): by ANOVA [^ in (A) to (C) and (E) and (F)], Benjamini-Yekutieli FDR [* in (D)], or t test [* in (H)]. (I) Time course of transcripts in IL-23/IL-36γ/OX40L–treated samples. n = 6. (J) Survival of MC38-S tumor– or MC38-R tumor–bearing animals treated intratumorally with mRNA and intraperitoneally with antibodies to deplete lymphocytes (n = 15). MC38-S control mRNA–treated alone, MC38-R with isotype control. Statistical significance by log-rank test (*; ns, not significant). Data are representative of three independent experiments (E to H; J, MC38-R), two independent experiments (A to C; J, MC38-S), or one independent experiment (D and I).

  • Fig. 5 Intratumoral delivery drives superior efficacy compared to other local routes of administration and inhibits tumor growth at distal sites.

    (A) Survival of MC38-R tumor–bearing animals treated with one or three 5-μg mRNA doses via intratumoral or peritumoral routes of administration (n = 15). Statistical significance by log-rank test (*) between intratumoral and subcutaneous/intradermal administration (solid lines, single dose; dashed lines, multiple dose). (B to D) Abscopal responses of bilateral MC38-S tumors treated once with 5 μg of total mRNA intratumorally into the right flank tumor only. Tumor volumes of both treated and untreated distal tumors (B, n = 20), time course of cytokines in tumors (C, n = 6), and transcripts in IL-23/IL-36γ/OX40L–treated samples (D, n = 6). (A to D) Five micrograms of control mRNA, 2.5 μg each of mRNA administered for doublets, and 1.67 μg each for triplet. Data are representative of two independent experiments (B) or one independent experiment (A, C, and D).

  • Fig. 6 Improved antitumor efficacy with combination of triplet mRNA therapy and checkpoint blockade.

    (A to C) Survival curves of MC38-R (A and B) or B16F10-AP3 (C) tumor-bearing mice after treatment with a single 5-μg dose of IL-23/IL-36γ/OX40L mRNA intratumorally with or without anti–PD-L1 (A and C) or anti–CTLA-4 (B) dosed intraperitoneally. Anti–PD-L1 and anti–CTLA-4 at 10 mg/kg per dose, twice per week for 2 weeks. Statistical significance indicated (*) by log-rank test (n = 15). Data are representative of two independent experiments (A and C) or one independent experiment (B).

  • Fig. 7 Response of human cells to IL-23, IL-36γ, and OX40L.

    (A to C) Dose-response curves of human PBMCs (A), MDMs (B), or MDDCs (C) to human IL-23 (A and B) or human IL-36γ (C), either recombinant or supernatant derived from mRNA-transfected cells. Secreted cytokines measured in the supernatant after 3 days (PBMCs) or 24 hours (MDMs and MDDCs). (D) Response of human CD4+ T cells to costimulation with human OX40L mRNA–transfected HeLa cells or mock-transfected cells. IL-2 was measured in the supernatant after 5 days. All data are representative of two independent experiments (two donors).

  • Table 1 Biodistribution of OX40L mRNA after intratumoral administration in MC38-R tumor–bearing mice.

    Quantification of modified mRNA after injection of 12.5 μg of OX40L mRNA. n = 3 animals per tissue (except n/a = not applicable, single sample was pooled from three animals).

    TissueTmax
    (hours)
    Cmax (ng/ml)AUC0-168 (ng*hour/ml)
    MeanSEMeanSE
    Tumor367802570406,000114,000
    Spleen2438894.314,8002920
    Proximal LNs6149n/a3000n/a
    Stomach322.220.9599306
    Distal LNs37.92n/a312n/a
    Lung36.465.0121193.1
    Kidney243.412.5814658.1
    Liver69.257.7513083.1
    Plasma33.242.9487.615.9
    Heart33.782.1670.946.6
    Brain241.881.0369.628.0

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/477/eaat9143/DC1

    Materials and Methods

    Fig. S1. In vitro OX40L bioactivity and in vivo cellular and tissue expression of OX40L after intratumoral mRNA administration.

    Fig. S2. Characterization of multiple syngeneic tumor models for responses to systemic checkpoint blockade therapies.

    Fig. S3. In vitro bioactivity of cytokine targets and in vivo expression of cytokine targets in tumor models.

    Fig. S4. Tolerability and dose-dependent efficacy of intratumoral mRNA therapy.

    Fig. S5. Induction of cytokines by multi-mRNA treatment.

    Fig. S6. Differentially expressed immune-related transcripts in control mRNA–treated tumors.

    Fig. S7. Activation status of DCs and T cells in response to local treatment with control mRNA.

    Fig. S8. Induction of granulocyte infiltration by multi-mRNA treatment and granulocyte contribution to antitumor efficacy.

    Fig. S9. DC profile in TdLN of mRNA-treated tumors and in vitro response of BMDCs to cytokines.

    Fig. S10. Induction of lymphocyte activation and remodeling of the TME by multi-mRNA treatment.

    Fig. S11. Overall transcript and costimulation focused transcript changes, and remodeling of the TME induced by multi-mRNA treatment.

    Fig. S12. Local mRNA therapy: Protein expression by alternate routes of administration, resistance to secondary tumor challenge, and antitumor memory.

    Fig. S13. Improved antitumor efficacy with combination of triplet mRNA and checkpoint blockade.

    Fig. S14. Response of human MDMs to IL-36γ.

    Table S1. Measurement of protein abundance in tumors after mRNA treatment and calculation of recombinant protein dosing.

    Table S2. Proteins, antibodies, and controls for in vivo dosing.

    Table S3. Probes in NanoString Plus Panel.

    Table S4. Flow cytometry staining antibodies.

    Data file S1. Primary data.

    Reference (55)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. In vitro OX40L bioactivity and in vivo cellular and tissue expression of OX40L after intratumoral mRNA administration.
    • Fig. S2. Characterization of multiple syngeneic tumor models for responses to systemic checkpoint blockade therapies.
    • Fig. S3. In vitro bioactivity of cytokine targets and in vivo expression of cytokine targets in tumor models.
    • Fig. S4. Tolerability and dose-dependent efficacy of intratumoral mRNA therapy.
    • Fig. S5. Induction of cytokines by multi-mRNA treatment.
    • Fig. S6. Differentially expressed immune-related transcripts in control mRNA–treated tumors.
    • Fig. S7. Activation status of DCs and T cells in response to local treatment with control mRNA.
    • Fig. S8. Induction of granulocyte infiltration by multi-mRNA treatment and granulocyte contribution to antitumor efficacy.
    • Fig. S9. DC profile in TdLN of mRNA-treated tumors and in vitro response of BMDCs to cytokines.
    • Fig. S10. Induction of lymphocyte activation and remodeling of the TME by multi-mRNA treatment.
    • Fig. S11. Overall transcript and costimulation focused transcript changes, and remodeling of the TME induced by multi-mRNA treatment.
    • Fig. S12. Local mRNA therapy: Protein expression by alternate routes of administration, resistance to secondary tumor challenge, and antitumor memory.
    • Fig. S13. Improved antitumor efficacy with combination of triplet mRNA and checkpoint blockade.
    • Fig. S14. Response of human MDMs to IL-36γ.
    • Table S1. Measurement of protein abundance in tumors after mRNA treatment and calculation of recombinant protein dosing.
    • Table S2. Proteins, antibodies, and controls for in vivo dosing.
    • Table S3. Probes in NanoString Plus Panel.
    • Table S4. Flow cytometry staining antibodies.
    • Reference (55)

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    Other Supplementary Material for this manuscript includes the following:

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