Direct targeting of FOXP3 in Tregs with AZD8701, a novel antisense oligonucleotide to relieve immunosuppression in cancer

Abstract

Background: The Regulatory T cell (Treg) lineage is defined by the transcription factor FOXP3, which controls immune-suppressive gene expression profiles. Tregs are often recruited in high frequencies to the tumor microenvironment where they can suppress antitumor immunity. We hypothesized that pharmacological inhibition of FOXP3 by systemically delivered, unformulated constrained ethyl-modified antisense oligonucleotides could modulate the activity of Tregs and augment antitumor immunity providing therapeutic benefit in cancer models and potentially in man.

Methods: We have identified murine Foxp3 antisense oligonucleotides (ASOs) and clinical candidate human FOXP3 ASO AZD8701. Pharmacology and biological effects of FOXP3 inhibitors on Treg function and antitumor immunity were tested in cultured Tregs and mouse syngeneic tumor models. Experiments were controlled by vehicle and non-targeting control ASO groups as well as by use of multiple independent FOXP3 ASOs. Statistical significance of biological effects was evaluated by one or two-way analysis of variance with multiple comparisons.

Results: AZD8701 demonstrated a dose-dependent knockdown of FOXP3 in primary Tregs, reduction of suppressive function and efficient target downregulation in humanized mice at clinically relevant doses. Surrogate murine FOXP3 ASO, which efficiently downregulated Foxp3 messenger RNA and protein levels in primary Tregs, reduced Treg suppressive function in immune suppression assays in vitro. FOXP3 ASO promoted more than 70% reduction in FOXP3 levels in Tregs in vitro and in vivo, strongly modulated Treg effector molecules (eg, ICOS, CTLA-4, CD25 and 4-1BB), and augmented CD8+ T cell activation and produced antitumor activity in syngeneic tumor models. The combination of FOXP3 ASOs with immune checkpoint blockade further enhanced antitumor efficacy.

Conclusions: Antisense inhibitors of FOXP3 offer a promising novel cancer immunotherapy approach. AZD8701 is being developed clinically as a first-in-class FOXP3 inhibitor for the treatment of cancer currently in Ph1a/b clinical trial (NCT04504669).

Keywords: Immunity, Cellular; Immunotherapy; Lymphocytes, Tumor-Infiltrating; Therapies, Investigational; Tumor Microenvironment.

Conflict of interest statement

Competing interests: The authors are paid employees of AstraZeneca or Ionis Pharmaceuticals, as indicated by their affiliations.

© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

AZD8701 is a highly potent clinical candidate ASO targeting human FOXP3. (A–C) Primary human Tregs were isolated from human PBMCs and cultured with AZD8701 or control ASO in duplicates for a total of 9 days, in the presence of Dynabeads human T-activated CD3/CD28 for the final 2 days of culture. (A) Line graph and (B) histogram show FOXP3 protein abundance in cultured Tregs as measured by flow cytometry. Histogram shows representative data for Tregs cultured with 1 µM ASO. (C) Histograms show the abundance of indicated proteins from a representative treatment with 1 µM AZD8701. (D–E) Contour plot and quantification of FOXP3 knockdown in human PBMC nTRegs and eTRegs with AZD8701 by flow cytometry. Data shown for three healthy donors. (F–G) iTregs were differentiated and cultured in the presence of ASOs in quadruplicates. Data represent ≥3 independent experiments and a total of ≥6 independent donors. (F) Line graph shows ability of iTregs to inhibit proliferation of effector cells in an in vitro suppression assay. (G) Histograms show representative CD25 or CellTrace Violet (CTV) staining on effector cells cultured at a 1:2 iTreg:Teffector ratio. (H) NSG mice were humanized by the infusion of human PBMC and treated systemically for four consecutive days with different AZD8701 doses. (I) FOXP3 messenger RNA expression was quantified by RT-qPCR. N=4 per group. (J) FOXP3 protein levels were quantified by flow cytometry in spleen, blood and bone marrow of humanized mice treated as in (F). N=7 per group. Data in figure is representative of ≥2 independent experiments. Error bars are ±SEM *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001 by one-way analysis of variance (ANOVA) with Dunnett’s post-test for E and J and two-way ANOVA with Dunnett’s post-test for F. Differences are calculated relative to control ASO (E, F and J) or saline (I). ASOs, antisense oligonucleotides; eTreg, effector Tregs; iTreg, inducible Tregs; nTreg, natural Tregs; PBS, phosphate buffered saline; Treg, regulatory T cells.

Figure 2

Antisense-mediated knockdown of mouse FOXP3 promotes loss of Treg suppressive phenotypic markers and function. (A) Primary Tregs were isolated from mouse spleen and cultured with ASOs (5 µM) in triplicates for 7 days. Bar chart shows relative FOXP3 mRNA expression measured by RT-qPCR, histogram shows FOXP3 protein expression measured by flow cytometry. (B) Inducible Tregs (iTregs) were differentiated and cultured in the presence of ASO for a total of 7 days. Heatmap shows expression of FOXP3-dependent mRNA genes as measured by Fluidigm. (C) Histograms show abundance of indicated proteins in primary Tregs. (D) iTregs were differentiated and cultured in the presence of ASOs and evaluated for their ability to inhibit proliferation of effector cells in an in vitro suppression assay in duplicates. Histograms show representative CellTrace Violet (CTV) staining on effector cells cultured at a 1:1 Treg:Teffector ratio. Data in figure represent ≥2 independent experiments. *, P≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001 by one-way analysis of variance (ANOVA) with Dunnett’s post-test for (A) and two-way ANOVA with Dunnett’s post-test for (D). Differences are calculated relative to control ASO. ASOs, antisense oligonucleotides; mRNA, messenger RNA; Tregs, regulatory T cells.

Figure 3

Mouse FOXP3 ASOs promote FOXP3 knockdown in vivo, which associates with phenotypes indicative of immunopotentiation. (A) Mice were systemically treated with FOXP3 ASOs for 3 weeks at 100 mg/kg/week dose. CD4 T cells were isolated from spleens and FOXP3 messenger RNA was measured by RT-qPCR. ASOs selected for further in vivo evaluation are highlighted in red and blue. N=4 per group. (B) iTregs were differentiated in the presence of ASO in duplicates for 5 days, and FOXP3 protein abundance was measured by flow cytometry. (C) Primary splenic nTregs were cultured in the presence of indicated ASOs in duplicates for 7 days, and FOXP3 protein abundance was measured by flow cytometry. (D–G) 20 mg/kg and 50 mg/kg FOXP3 ASOs or 50 mg/kg control ASO were administered to BALB/c mice via subcutaneously route 5 days on 2 days off. N=5 per group. (D) The line graph shows the frequency of gated FOXP3+ cells within the splenic CD4+ population. Pseudocolor density plots show representative FOXP3+ gating. (E) Line graph shows programmed cell death 1 protein expression on detectable FOXP3+ cells. (F) Line graphs show the frequency of CD62LloCD44hi T-memory cells within the total CD4+ T-cell population. (G) Scatter bar charts show the frequency of CD69+ cells within the CD4+ or CD8+ populations at the week six time point. (H) ASOs were dosed (100 mg/kg two times per week) for 3 weeks before spleens from cohorts of mice were analyzed by flow cytometry at indicated time points following cessation of treatment. N=5 per group. Data represent two independent experiments. Error bars are SEM data in figure represent ≥2 independent experiments. *, P≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001 by one-way analysis of variance (ANOVA) with Dunnett’s post-test for (A and G) and two-way ANOVA with Dunnett’s post-test for (D, E, F and H). Differences are calculated relative to control ASO (D–H) or saline (A). ASOs, antisense oligonucleotides; iTreg, inducible Tregs; nTreg, natural Tregs; Tregs, regulatory T cells.

Figure 5

FOXP3 ASOs promote antitumor efficacy when combined with αPD-L1 immune checkpoint blockade. (A) Mice were treated with mouse FOXP3 ASO (895317) (50 mg/kg BIW) and αPD-L1 (10 mg/kg BIW) alone or in combination from day 1 post A20 tumor-implantation and dosed two times per week for the duration of the experiment. N=14 per group. (B) Mean tumor and (C) individual tumor volumes and indicated number of complete responses (CR). (D-E) Tumor samples and peripheral blood (PB) were analyzed at day 20 by flow cytometry and RNA levels, respectively. (D) Frequency of CD8 +T cells and dendritic cells in tumor and (E) cytotoxic T cell and antigen-presenting cell gene markers in PB. *, P≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001 by one-way analysis of variance (ANOVA) with Dunnett’s post-test for (D and E) and two-way ANOVA with Dunnett’s post-test for (B). Differences are calculated relative to PBS. ASOs, antisense oligonucleotides; BIW, two times per week; PBS, phosphate buffered saline; PD-1, programmed cell death 1; PD-L1, programmed cell death ligand 1.

Figure 4

Single-agent antitumor efficacy mediated by mouse FOXP3 ASOs. (A–F) ID8-VEGF or A20 tumor-bearing mice were systemically treated with FOXP3 ASOs starting at day 1 after implant at 50 mg/kg BIW until day 62 (ID8-VEGF) or four times per week until day 24 (A20). N=8 per group. (A) Tumors were dissociated and tumor-infiltrating Tregs were analyzed by flow cytometry. Helios +CD4 Tregs are shown in red. (B) FOXP3 protein expression was measured in Helios +CD4 Tregs. Line graphs show (C) ID8-VEGF and (E) A20 tumor volumes. The number of complete responses (CR) vs total number of animals in the group is indicated next to lines. (D) ID8-VEGF tumors were dissociated, and abundance of total tumor-infiltrating leukocytes (CD45 +cells) and CTL (CD8 +T cells) were analyzed by flow cytometry. (F) Total RNA from A20 tumors was analyzed for the mRNA expression of CTL marker CD8 and immune cell activation marker GzmB. (G) A20 tumor-bearing mice were treated with indicated ASO or vehicle control 50 mg/kg BIW and tumors analyzed by flow cytometry at day 13 or day 20 time points. N=10 per group. Bar charts show tumor or spleen Foxp3 and GzmB expression measured by RT-qPCR. (H) CD8+ T cells were depleted in vivo with an αCD8 blocking antibody in A20 tumor-bearing mice that were treated with indicated ASO or control at 50 mg/kg BIW. N=12 per group. Data represent >4 independent experiments in (A–F), two independent experiments in (G) and a single cell depletion experiments in (H). *, P≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.000 by one-way analysis of variance (ANOVA) with Dunnett’s post-test for (B, D, F and G) and two-way ANOVA with Dunnett’s post-test for (C, E and H). Differences are calculated relative to PBS (B–G) or as indicated on the panel (H). ASOs, antisense oligonucleotides; BIW, two times per week; mRNA, messenger RNA; PBS, phosphate buffered saline; VEGF, vascular endothelial growth factor.

Figure 6

FOXP3 ASOs reprogram Treg effector cell phenotype in vivo in combination with αPD-L1 immune checkpoint blockade. (A) Mice were treated with ASO (50 mg/kg BIW) and αPD-L1 (10 mg/kg BIW) alone or in combination from day 1 post A20 tumor-implantation. N=14 per group. Tumors were analyzed by mass cytometry at day 20 after implantation. (B) t-SNE analysis of tumor-infiltrating CD4+ T cells. (C) Relative changes of CD4 +T cell clusters compared with control group. (D) Expression map of T cells and Tregs markers. (E) Quantification of Treg activation expression markers. Error bars are SD *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001 by one-way analysis of variance with Dunnett’s post-test relative to control ASO. ASOs, antisense oligonucleotides; BIW, two times per week; t-SNE, t-distributed stochastic neighbor embedding; PD-L1, programmed cell death ligand 1; Treg, Regulatory T cells.

Figure 7

FOXP3 ASOs promote additive efficacy when combined with αPD-1 immune checkpoint blockade. Mice were treated with ASO 895310 (50 mg/kg BIW) and αPD-1 (10 mg/kg BIW) alone or in combination from day 1 post A20 tumor-implantation. ASO was dosed for the duration of the study. αPD-1 was dosed six times. N=12 per group. Mice were sacrificed when tumors exceeded 1500 mm3 or after 40 days. (A) Spider plots indicate tumor volumes. Survival panel shows percent surviving mice vs time. (B–G) Foxp3, CD8a/CD8b and Granzyme messenger RNA expression in tumors and spleens from terminal samples. (H) Model of ASO targeting of FOXP3 expression to reduce Treg immunosuppressive capacity and promote antitumor immunity. Data in figure represent two independent experiments. CR, complete responses. Error bars are ±SEM *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001 by one-way analysis of variance with Dunnett’s post-test relative to PBS group. Survival analysis (A) was done by log-rank Mantel-Cox test. ASOs, antisense oligonucleotides; BIW, two times per week; IFN, interferon; PBS, phosphate buffered saline; PD-1, programmed cell death 1.