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Recruitment of CD103+ dendritic cells via tumor-targeted chemokine delivery enhances efficacy of checkpoint inhibitor immunotherapy

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Science Advances  11 Dec 2019:
Vol. 5, no. 12, eaay1357
DOI: 10.1126/sciadv.aay1357
  • Fig. 1 CBD-CCL4 exhibits high affinity to collagen and accumulates in tumor following intravenous injection.

    (A) WT CCL4 and CBD-CCL4 were analyzed by SDS-PAGE followed by Coomassie blue staining. (B and C) Affinity of CBD-CCL4 against (B) collagen I and (C) collagen III was measured by SPR. SPR chips were functionalized with collagen I [~500 resonance units (RU)] and collagen III (~700 RU), and CBD-CCL4 was flowed over the chips at indicated concentrations. Curves represent the obtained specific responses (in resonance units) to CBD-CCL4. Experimental curves were fitted with 1:1 Langmuir fit model. Binding kinetics values [dissociation constants (Kd) and rate constants (kon and koff)] determined from the fitted curves are shown. (D and E) Binding of (D) WT CCL4 or (E) CBD-CCL4 to human melanoma cryosections as determined by immunofluorescence microscopy. Scale bars, 100 μM. DAPI, 4′,6-diamidino-2-phenylindole. (F) GPCR activation assay comparing signaling of WT CCL4 and CBD-CCL4 in THP1 monocytes. Median effective concentration (EC50) values were calculated using a nonlinear dose-response curve fit model. Each point represents mean ± SEM, n = 3. (G) Blood plasma pharmacokinetics was analyzed using DyLight 800–labeled WT CCL4 or CBD-CCL4 in B16F10 melanoma. Four days after tumor inoculation, mice were administered 25 μg of WT CCL4 or the molar equivalent of CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4) via intravenous injection. Blood was collected at the indicated time points, and plasma was separated and analyzed for CCL4 concentration. Each point represents mean ± SEM, n = 4. (H) Biodistribution was analyzed using DyLight 647–labeled WT CCL4 or CBD-CCL4 in EMT6 breast cancer. When the tumor volume reached 500 mm3, 25 μg of WT CCL4 or the molar equivalent of CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4) was given via intravenous injection. Fluorescence intensity in each tumor was measured using an in vivo imaging system (IVIS), converted to percent injected dose using a known standard series, and normalized to the weight of the tumor. Each bar represents mean ± SEM, n = 3. **P < 0.01.

  • Fig. 2 CBD-CCL4 fusion recruits DCs and T cells and improves efficacy of CPI therapy in B16F10 melanoma.

    Mice were intradermally injected with 5 × 105 cells; 4 days later, mice were treated with WT CCL4 (25 μg given via intravenous injection) or molar equivalent CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4, given via intravenous injection) in combination with CPI antibody therapy consisting of αPD-L1 and αCTLA4 (100 μg each) given via intraperitoneal injection. CPI therapy alone was administered as control. (A) Tumor growth was monitored over time until 10 days after tumor inoculation, at which point tumors were harvested and processed for flow cytometry analysis. (B to H) Immune cell composition was evaluated, where graphs depict the number of (B) CD45+ leukocytes, (C) CD103+ CD11c+ MHCIIHi DCs, (D) total CD11c+ DCs, (E) CD8+ T cells, (F) NK1.1+ CD3 NK cells, (G) CD4+ T cells, and (H) % FoxP3+ CD25+ Tregs (of total CD4+ T cells). Bars represent means ± SEM, n = 11 to 13. *P < 0.05 and **P < 0.01. Arrow in (A) indicates time of treatment. (I to N) Regression analysis comparing the number of tumor-infiltrating cells with tumor volume was performed using the results obtained in (A) to (H). Correlations between (I) tumor volume and CD103+ CD11c+ MHCIIHi DCs, (J) tumor volume and CD8+ T cells, (K) CD103+ CD11c+ MHCIIHi DCs and CD8+ T cells, (L) tumor volume and NK1.1+ CD3 NK cells, (M) tumor volume and total CD11c+ DCs, and (N) tumor volume and total CD45+ leukocytes.

  • Fig. 3 CBD-CCL4 combination treatment recruits cross-presenting DCs and T cells and improves efficacy of CPI therapy in EMT6 immune-excluded breast cancer.

    Mice were subcutaneously injected with 5 × 105 cells; 6 and 9 days after inoculation, the mice were treated with WT CCL4 (25 μg given via intravenous injection) or molar equivalent CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4, given via intravenous injection) in combination with CPI antibody therapy consisting of αPD-L1 and αCTLA4 (100 μg each) given via intraperitoneal injection. CPI therapy alone was administered as control. (A) Tumor growth was monitored over time until 10 days after tumor inoculation, at which point tumors were harvested and processed for flow cytometry analysis. (B to H) Immune cell composition was evaluated, where graphs depict the number of (B) CD45+ leukocytes, (C) CD103+ CD11c+ MHCIIHi DCs, (D) CD8α+ CD11c+ MHCIIHi DCs, (E) total CD11c+ DCs, (F) CD8+ T cells, (G) CD4+ T cells, and (H) % FoxP3+ CD25+ Tregs (of total CD4+ T cells). Bars represent means ± SEM, n = 7 to 9. *P < 0.05 and **P < 0.01 Arrows indicate time of treatment.

  • Fig. 4 CBD-CCL4 therapy does not elevate treatment-related adverse events.

    Mice were intradermally injected with 5 × 105 cells; 4 and 7 days later, the mice were treated with WT CCL4 (25 μg given via intravenous injection) or molar equivalent CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4, given via intravenous injection) in combination with CPI antibody therapy consisting of αPD-L1 and αCTLA4 (100 μg each) given via intraperitoneal injection. CPI therapy alone was administered as control. (A) ALT activity in serum of mice, relative to saline-treated control mice, as measured 10 days after tumor inoculation. (B and C) Eight days after tumor inoculation, blood was collected, and serum levels of (B) IFN-γ and (C) IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). (D) Ten days after tumor inoculation, the lung, kidney, and liver were harvested, and histological analysis was performed to assess tissue morphology and immune cell infiltration. Representative images of each organ are shown. Scale bars, 200 μm. All bars represent means ± SEM, n = 6 to 8.

  • Fig. 5 CBD-CCL4 requires Batf3-lineage DCs and mediates downstream effector T cell recruitment.

    (A to G) Batf3−/− mice were intradermally injected with 5 × 105 B16F10 cells; 7 days later, once tumor volume exceeded 50 mm3, the mice were treated with CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4, given via intravenous injection) in combination with CPI antibody therapy consisting of αPD-L1 and αCTLA4 (100 μg each) given via intraperitoneal injection. CPI therapy alone was administered as comparison. Graphs display (A) tumor growth curves and (B to G) tumor immune infiltrates depicting the number of (B) CD45+ leukocytes, (C) total CD11c+ DCs, (D) CD103+ CD11c+ MHCIIHi DCs, (E) CD8+ T cells, (F) CD4+ T cells, and (G) NK cells. Graphs depict means ± SEM, n = 6. **P < 0.01. (H and I) C57BL/6 mice were intradermally injected with 5 × 105 B16F10 cells; 7 days later, the mice were treated with CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4, given via intravenous injection) in combination with CPI antibody therapy consisting of αPD-L1 and αCTLA4 (100 μg each) given via intraperitoneal injection, as shown with a black arrow. CPI therapy alone was administered as comparison. In addition, indicated groups were treated with 200 μg of CXCR3 blocking antibody via intraperitoneal injection on days 7, 9, and 11, as shown by blue arrows. (H) Tumor growth curves and (I) survival curves are shown, with each bar representing mean ± SEM, n = 5. (J) C57BL/6 mice were intradermally injected with 5 × 105 B16F10 cells; 4 days later, the mice were treated with saline, CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4, given via intravenous injection) or CPI antibody therapy consisting of αPD-L1 and αCTLA4 (100 μg each) given via intraperitoneal injection. Tumor growth curves are shown, with each bar representing mean ± SEM, n = 5. **P < 0.01. Arrows indicate time of treatment.

  • Fig. 6 CBD-CCL4 in combination with CPI slows growth of implantable and spontaneous MMTV-PyMT breast tumors.

    (A to F) 106 MMTV-PyMT cells were inoculated on the right mammary fat pad. Six days after inoculation, mice were treated with CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4, given via intravenous injection) in combination with CPI antibody therapy consisting of αPD-L1 and αCTLA4 (100 μg each) given via intraperitoneal injection. CPI therapy alone was administered as comparison. (A) Tumor growth curves and (B) survival curves are shown, with each bar representing mean ± SEM, n = 9 to 10. Numbers indicate the fraction of tumor-free mice in each group. *P < 0.05 and **P < 0.01. (C) One month after becoming tumor free, the mice were given a secondary tumor challenge with 106 MMTV-PyMT cells in the contralateral mammary fat pad. Naïve control mice were inoculated in a similar fashion. Numbers indicate how many mice remain tumor free 14 days following tumor challenge. (D to F) Twenty days after tumor rechallenge, the mice were euthanized, and splenocytes were stimulated with PMA and ionomycin for 6 hours to evaluate cytokine production. (D) Charts depict average percentage of splenic CD8+ T cells and CD4+ T cells producing the indicated cytokines. *P < 0.05. (E and F) Percentage of IFN-γ–producing effector (CD44+) (E) CD8+ T cells and (F) CD4+ T cells in the spleen. Bars represent means ± SEM, n = 5. *P < 0.05. (G) Spontaneous MMTV-PyMT tumor-bearing mice were monitored until total tumor burden reached 100 mm3. At this point, the mice were treated with CBD-CCL4 (25 μg of CCL4 basis or 93 μg of CBD-CCL4, given via intravenous injection) in combination with CPI antibody therapy consisting of αPD-L1 and αCTLA4 (100 μg each) given via intraperitoneal injection. CPI therapy alone was administered as comparison. Identical dosing was given 7 and 14 days after the initial treatment. Tumor growth curves until the first mouse died are shown. Graphs depict means ± SEM, n = 6. *P < 0.05 and **P < 0.01. Arrows indicate time of treatment.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/12/eaay1357/DC1

    Fig. S1. Dynamic light scattering measurement of CBD-CCL4.

    Fig. S2. Binding of CBD-CCL4 and WT CCL4 to collagen and murine melanoma sections.

    Fig. S3. In vivo imaging of EMT6 tumors.

    Fig. S4. Representative flow cytometry gating strategy.

    Fig. S5. Cell infiltrate analysis of effector T cells, MDSCs, and macrophages in B16F10 melanoma.

    Fig. S6. Immunofluorescence analysis of CD8+ cells and CD11c+ DCs in EMT6 breast cancer.

    Fig. S7. Tumor growth curves of CT26 and MC38 following treatment with anti–PD-1 + CBD-CCL4.

    Fig. S8. CCR5 expression on T cells and DCs.

    Fig. S9. Survival curves of spontaneous MMTV-PyMT mice following treatment.

    Table S1. Sequences of CCL4, CBD protein, and CBD-CCL4 fusion protein.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Dynamic light scattering measurement of CBD-CCL4.
    • Fig. S2. Binding of CBD-CCL4 and WT CCL4 to collagen and murine melanoma sections.
    • Fig. S3. In vivo imaging of EMT6 tumors.
    • Fig. S4. Representative flow cytometry gating strategy.
    • Fig. S5. Cell infiltrate analysis of effector T cells, MDSCs, and macrophages in B16F10 melanoma.
    • Fig. S6. Immunofluorescence analysis of CD8+ cells and CD11c+ DCs in EMT6 breast cancer.
    • Fig. S7. Tumor growth curves of CT26 and MC38 following treatment with anti–PD-1 + CBD-CCL4.
    • Fig. S8. CCR5 expression on T cells and DCs.
    • Fig. S9. Survival curves of spontaneous MMTV-PyMT mice following treatment.
    • Table S1. Sequences of CCL4, CBD protein, and CBD-CCL4 fusion protein.

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