Intratumoral STING Activation with T-cell Checkpoint Modulation Generates Systemic Antitumor Immunity

Abstract

Coordinated manipulation of independent immune regulatory pathways in the tumor microenvironment-including blockade of T-cell checkpoint receptors and reversal of suppressive myeloid programs-can render aggressive cancers susceptible to immune rejection. Elevated toxicity associated with combination immunotherapy, however, prevents translation of the most efficacious regimens. We evaluated T-cell checkpoint-modulating antibodies targeting CTLA-4, PD-1, and 4-1BB together with myeloid agonists targeting either STING or Flt3 in the TRAMP-C2 model of prostate cancer to determine whether low-dose intratumoral delivery of these agents could elicit systemic control of multifocal disease. Intratumoral administration of the STING agonist cyclic di-GMP (CDG) or Flt3 Ligand (Flt3L) augmented the therapeutic effect of systemic triple checkpoint modulation and promoted the cure of 75% of mice with bilateral TRAMP-C2; however, when all agents were administered locally, only CDG mobilized abscopal immunity. Combination efficacy correlated with globally enhanced ratios of CD8⁺ T cells to regulatory T cells (Treg), macrophages, and myeloid-derived suppressor cells, and downregulation of the M2 marker CD206 on tumor-associated macrophages. Flt3L improved CD8⁺ T-cell and dendritic cell infiltration of tumors, but was diminished in efficacy by concomitant Treg expansion. Although intratumoral CDG/checkpoint therapy invokes substantial ulceration at the injection site, reduced CDG dosing can preserve tissue integrity without sacrificing therapeutic benefit. For high-order combinations of T-cell checkpoint antibodies and local myeloid agonists, systemic antibody administration provides the greatest efficacy; however, local administration of CDG and antibody provides substantial systemic benefit while minimizing the potential for immune-related adverse events. Cancer Immunol Res; 5(8); 676-84. ©2017 AACR.

Figure 1. Local CDG potentiates IP checkpoint modulation against bilateral TRAMP-C2

(A) Five to eight-week-old C57BL/6 mice were challenged with 1×10⁶ TRAMP-C2 cells on both flanks, and were treated on days 14, 18, and 22 post-implantation with intraperitoneal (IP) checkpoint modulators and/or intratumoral (IT) CDG at either the right (R) or both right and left flank tumors (R&L). (B) Percent of mice tumor free at each flank, (C) tumor growth kinetics, and (D) overall survival over the treatment period are shown. Mice were deemed moribund when tumor volume reached 1000 mm³. Data is cumulative of n = 3 experiments with 5 mice per group. Statistical significance for survival was calculated using the Gehan-Breslow-Wilcoxon test. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Figure 2. Local Flt3L potentiates IP checkpoint modulation against bilateral TRAMP-C2

(A) Five to eight-week-old C57BL/6 mice were challenged with 1×10⁶ TRAMP-C2 cells on both flanks, and were treated on days 14, 18, and 22 post-implantation with intraperitoneal (IP) checkpoint modulators and/or intratumoral (IT) Flt3L at either the right (R) or both right and left flank tumors (R&L). (B) Percent of mice tumor free at each flank, (C) tumor growth kinetics, and (D) overall survival over the treatment period are shown. Mice were deemed moribund when tumor volume reached 1000 mm³. Data is cumulative of n = 3 experiments with 5 mice per group. Statistical significance for survival was calculated using the Gehan-Breslow-Wilcoxon test. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Figure 3. Intratumoral combination CDG and checkpoint modulation mobilizes abscopal responses against distal TRAMP-C2

(A) Five to eight-week-old C57BL/6 mice were challenged with 1×10⁶ TRAMP-C2 cells on both flanks, and were treated on days 14, 18, and 22 post-implantation with intratumoral CDG and/or checkpoint modulators at the right flank tumor only. (B) Percent of mice tumor free at each flank, (C) tumor growth kinetics, and (D) overall survival over the treatment period are shown. Mice were deemed moribund when tumor volume reached 1000 mm³. Data is cumulative of n = 3–4 experiments with 5–10 mice per group. Statistical significance for survival was calculated using the Gehan-Breslow-Wilcoxon test. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Figure 4. Intratumoral CDG with checkpoint modulation invokes dose-dependent and tumor size-dependent ulcerative pathology at the tumor site

(A) Photographs of representative mice treated as described in Fig. 3 were taken six days following final intratumoral injection (Day 28), when ulceration is most intense, and two weeks later during the resolution phase (Day 42). (B) Mice bearing either 14-day or 28-day established TRAMP-C2 tumors were given three intratumoral injections of CDG with anti-CTLA-4, anti-PD-1, and anti-4-1BB every four days for a total of three injections, according to normal protocol. Photographs of ulceration were taken 2 (Day 24) and 6 (Day 28) days following final intratumoral injection.

Figure 5. Intratumoral combination Flt3L and checkpoint modulation does not mobilize abscopal responses against distal TRAMP-C2

(A) Five to eight-week-old C57BL/6 mice were challenged with 1×10⁶ TRAMP-C2 cells on both flanks, and were treated on days 14, 18, and 22 post-implantation with intratumoral Flt3L and/or checkpoint modulators in the right flank only. (B) Percent of mice tumor free at each flank, (C) tumor growth kinetics, and (D) overall survival over the treatment period are shown. Mice were deemed moribund when tumor volume reached 1000 mm³. Data is cumulative of n = 3–4 experiments with 5 mice per group. Statistical significance for survival was calculated using the Gehan-Breslow-Wilcoxon test. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Figure 6. Analysis of the TRAMP-C2 immune infiltrate at local and distal tumors following intratumoral therapy at a single lesion

(A) To obtain cell numbers sufficient for flow analysis, TRAMP-C2 tumors were established for 31 days, exposed to three doses of intratumoral therapy at the right flank, then were harvested for multi-parameter flow cytometry as described in Methods. See Supplementary materials for antibodies used and gating strategy. (B) Relative proportions of unique cell subsets within the CD45⁺ immune infiltrate, normalized to the total number of gated cells. (C) Relative proportions of T-cell subsets within the CD3⁺ lymphocyte infiltrate, normalized to the total number of gated T cells. (D) Relative proportions of MDSC subsets within the CD11b⁺Gr-1⁺ immune infiltrate. (E) Calculated ratios of CD8⁺ T cells to Treg, (F) density of SPAS-1 tetramer⁺ CD8⁺ T-cells, (G) percentage of SPAS-1 reactive T cells within all infiltrating CD8 T cells, and ratio of CD8⁺ T cells to (H) macrophages are shown. (I) Percentage of macrophages expressing the M2 macrophage marker CD206. Data is cumulative of 2 independent experiments with 5 mice per group. Statistical significance was calculated using Student’s T test. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.