Tumor Regression upon Intratumoral and Subcutaneous Dosing of the STING Agonist ALG-031048 in Mouse Efficacy Models

Abstract

Stimulator of interferon genes (STING) agonists have shown potent anti-tumor efficacy in various mouse tumor models and have the potential to overcome resistance to immune checkpoint inhibitors (ICI) by linking the innate and acquired immune systems. First-generation STING agonists are administered intratumorally; however, a systemic delivery route would greatly expand the clinical use of STING agonists. Biochemical and cell-based experiments, as well as syngeneic mouse efficacy models, were used to demonstrate the anti-tumoral activity of ALG-031048, a novel STING agonist. In vitro, ALG-031048 is highly stable in plasma and liver microsomes and is resistant to degradation via phosphodiesterases. The high stability in biological matrices translated to good cellular potency in a HEK 293 STING R232 reporter assay, efficient activation and maturation of primary human dendritic cells and monocytes, as well as long-lasting, antigen-specific anti-tumor activity in up to 90% of animals in the CT26 mouse colon carcinoma model. Significant reductions in tumor growth were observed in two syngeneic mouse tumor models following subcutaneous administration. Combinations of ALG-031048 and ICIs further enhanced the in vivo anti-tumor activity. This initial demonstration of anti-tumor activity after systemic administration of ALG-031048 warrants further investigation, while the combination of systemically administered ALG-031048 with ICIs offers an attractive approach to overcome key limitations of ICIs in the clinic.

Keywords: STING agonist; immune checkpoint inhibitor; immuno-oncology; syngeneic mouse model.

Figure 1

Chemical Structures of 2′3′ cGAMP, ADU-S100, and ALG-031048. Chemical modifications in ALG-031048 relative to 2′3′ cGAMP are highlighted in red.

Figure 2

In vitro STING R232 activation of 2′3′ cGAMP, ADU-S100, and ALG-031048 in a biochemical thermal shift assay (A), as well as in HEK 293 R232 cells with an IRF (B) and IFN-ß reporter (C). Shown are the mean of three biological replicates ± SD as error bars of representative dose–response curves.

Figure 3

Upregulation of CD83 (A,C) and CD86 (B,D) on the surface of primary human immature dendritic cells (A,B) and monocytes (C,D). Monocytes were treated for 24 h, while immature dendritic cells were treated for 72 h. Control cells unstained for CD83 and CD86, respectively, shaded in gray, unstimulated cells (dashed black line), and cells stimulated with 200 μM of ALG-031048 (blue line). The gating strategy is provided in Supplementary Figure S3.

Figure 4

Tumor regression in CT26-tumor-bearing BALB/c mice upon IT treatment of 25 μg (A, light red) or 100 μg of ADU-S100 (B, dark red) or 25 μg (C, light blue) or 100 μg of ALG-031048 (D, dark blue) on Days 1, 4, and 7 as indicated with the upward tick marks. Vehicle-treated animals are shown in gray; each line depicts the tumor volume over time of one animal, and there are 10 animals in each group. Treatment started when tumors reached a median volume of 116 mm³, 10 days post-implantation.

Figure 5

(A) ALG-031048 provides long-lasting anti-tumor activity in vivo. Nine CT26-tumor-bearing BALB/c mice, which showed complete tumor regression in Figure 4 after treatment with ALG-031048, were re-challenged with an SC injection of CT26 on the contra-lateral flank. Ten naïve, age-matched mice served as controls. Tumor volume was measured every 3 days. Each line depicts the tumor volume over time in one animal. p-value of <0.001 for ALG-031048 pretreated vs. naïve animals based on time to endpoint in days. (B) Complete tumor suppression in CT26-bearing BALB/c mice treated with three IT doses of 100 μg of ALG-031048 (blue) compared with vehicle-treated animals (gray). p-value of <0.001 for ALG-031048 vs. vehicle based on time to endpoint in days. Treatment started at a median tumor volume of 108 mm³, 14 days after implantation. These animals (blue) were next re-challenged with CT26 (C) and EMT-6 (D), and tumor growth was observed for 25 days without additional therapeutic intervention; naïve animals were used as controls (gray). p-value of <0.001 for ALG-031048 vs. vehicle treatment for the CT26 tumors (C) and p-value of 0.125 (non-significant) for the EMT-6 tumors (D), based on tumor volume on the last available measurement.

Figure 6

(A) Improved anti-tumor activity of CT26-tumor-bearing mice with the combination treatment of anti-CTLA-4 and ALG-031048. CT26 tumor growth curves of animals treated with vehicle (gray) or 25 μg of ALG-031048 dosed intratumorally on Days 1, 4, and 7 (A, light blue), or 5 mg/kg anti-CTLA-4 on Day 1, followed by 1 mg/kg anti-CTLA-4 on Days 4 and 7 (B, red), or a combination of ALG-031048 and anti-CTLA-4 (C, green). Upward tick marks on Days 1, 4, and 7 indicate times of treatment. Treatment started at a median tumor volume of 108 mm³, 13 days post-implantation. Tumor volumes were measured every 3 days. Each line depicts the tumor volume over time of one animal, with ten animals in each group.

Figure 7

(A,B): In vivo efficacy after SC dosing of ALG-031048. 10 CT26-tumor-bearing mice per group were treated with SC with 1 (A, dark blue) or 4 mg/kg ALG-031048 (B, dark green), or IT with 25 μg (A, light blue) or 100 μg of ALG-031048. Vehicle-treated control animals are shown in gray. All animals received three treatments on Days 1, 4, and 7, as indicated with the upward tick marks, starting at a mean tumor volume of 108 mm³ at 13 days post-implantation. (C–E) Anti-tumor activity of ALG-031048-dosed SC in the MC38-hPD-L1 mouse tumor model. A total of 10 female MC38-hPD-L1 tumor-bearing C57BL/6 mice per group were treated with 4 BIW doses of 5 mg/kg atezolizumab IP on Days 1, 4, 8, and 11 as indicated with upward tick marks (C, red), 2 QW doses of 0.5 mg/kg ALG-031048 SC on Days 1 and 8 as indicated with downward tick marks (D, blue), or a combination of ALG-031048 SC and atezolizumab IP (E, purple). Vehicle-treated control animals are shown in gray. Treatment was initiated on Day 1 post-implantation, at a median tumor volume of 69 mm³.