PEGylated arginine deiminase can modulate tumor immune microenvironment by affecting immune checkpoint expression, decreasing regulatory T cell accumulation and inducing tumor T cell infiltration

Abstract

PEGylated arginine deiminase (ADI-PEG 20) is being investigated in clinical studies in arginine auxotrophic cancers and is well-tolerated. The anti-tumor properties of ADI-PEG 20 have been extensively investigated - ADI-PEG 20 inhibits the growth of auxotrophic cancers in vitro and in vivo - however, its impact on immune cells is largely unknown. Here we report the potential impact of ADI-PEG 20 on the tumor immune microenvironment. ADI-PEG 20 induced immunosuppressive programmed death-ligand 1 expression on some cancer cells in vitro, but the magnitude of the increase was cell line dependent and in most relatively small. Using healthy donor human peripheral blood mononuclear cells (PBMCs) we discovered that when present during initiation of T cell activation (but not later on) ADI-PEG 20 can inhibit their differentiation after early activation stage manifested by the expression of CD69 marker. In vivo, ADI-PEG 20 induced tumor T-cell infiltration in a poorly immunogenic syngeneic mouse melanoma B16-F10 model and reduced its growth as a single agent or when combined with anti-PD-1 mAb. It was also effective by itself or in combination with anti-PD-L1 mAb in CT26 colon carcinoma syngeneic model.

Keywords: PD-L; TILs; Treg; arginine deiminase; immunomodulation.

Figure 1. Effect of ADI-PEG 20 on PD-L1 cell surface levels in a cancer cell panel

Cancer cells were treated with serially diluted ADI-PEG 20 or IFNγ control and their surface PD-L1 was assessed by flow cytometry at 24 h (A), 48 h (B) and 72 h (C) after the start of the treatment.

Figure 2. Effect of ADI-PEG 20 on PD-L1 expression in a cancer cell panel

Cancer cells were treated with serially diluted ADI-PEG 20 or IFNγ control and PD-L1 mRNA levels were measured by RT-qPCR at 24 h (A), 48 h (B) and 72 h (C) after the start of the treatment. β-actin mRNA was used for normalization.

Figure 3. ADI-PEG 20 treatment during PBMC stimulation and not under resting conditions lead to a sustained increase in CD69+ T cells

PBMCs were stimulated with anti-CD3/CD28 Dynabeads in the presence or absence of ADI-PEG 20 or mutant ADI-PEG 20. Percentages of CD69+ cells among CD4+ T cells (A) and CD8+ T cells (B) were determined by flow cytometry at 24 h, 48 h & 72 h. PBMCs containing CD69+ T cells in the absence of stimulation were not affected by 20 nM ADI-PEG 20 or mutant ADI-PEG 20 (C). NT – non-treated.

Figure 4. CD25+ T cell induction was affected by high (and not low) concentrations of ADI-PEG 20 when added during PBMC stimulation and not under resting conditions or when added 48 h after the addition of anti-CD3/CD28 Dynabeads

Figure 5

IL-2 (A) and IFNγ (B) secretion by PBMCs after stimulation with anti-CD3/CD28 Dynabeads in the presence or absence of ADI-PEG 20 or mutant ADI-PEG 20. ADI-PEG 20 was added simultaneously with the beads or 24 h or 48 h later. IL2 and IFNγ in the media were measured by ELISA at 24 h, 48 h and 72 h after initiation of the stimulation.

Figure 6. ADI-PEG 20 blocks anti-CD3/CD28 induced upregulation of PD-1, CTLA-4 and PD-L1 on T cells

PBMCs were stimulated with anti-CD3/CD28 Dynabeads in the presence or absence of ADI-PEG 20 or mutant ADI-PEG 20. Percentages of PD-1+ (A–B), CTLA-4+ (C–D) and PD-L1+ (E–F) cells in CD4+ T cells (A, C, E) and CD8+ T cells (B, D, F) were determined by flow cytometry at 24 h, 48 h & 72 h.

Figure 7. ADI-PEG 20 inhibits accumulation or T cells with Treg markers

PBMCs were stimulated with anti-CD3/CD28 Dynabeads in the presence or absence of ADI-PEG 20 or mutant ADI-PEG 20. Percentages of Treg cells among CD4+ T cells were determined by flow cytometry at 24 h, 48 h & 72 h. Treg cells were determined by CD3+CD4+CD25+FoxP3+ (A), CD3+CD4+CD25+FoxP3+PD-L1+ (B) or CD3+CD4+CD25+FoxP3+CTLA-4+ (C) markers.

Figure 8. ADI-PEG 20 induces T cell infiltration into B16-F10 tumors

B16-F10 cells were implanted into C57BL/6 mice and the mice were treated IM with 12 mg/kg of ADI-PEG 20 or vehicle (PBS) control on d1 and d7 post tumor implantation. On day 14 tumors were removed and sectioned. Sections were stained by IHC with anti-CD3 mAb and counterstained with DAPI. Number of T cells per field at 400× was counted in four sections of each tumor and averaged (A), representative images taken at 400× magnification (B). One-way Anova was used for statistical analysis, ns – non significant, ****p < 0.0001.

Figure 9. ADI-PEG 20 reduces B16-F10 tumor growth as a single agent and in combination with anti-mPD-1 mAb

B16-F10 cells were implanted into C57BL/6J mice and the next day the mice were treated with 12 mg/kg of ADI-PEG 20 QWx2 IM, 10 mg/kg anti-mPD-1 Q2D IP, 12 mg/kg of ADI-PEG 20 QWx2 IM and 10 mg/kg anti-mPD-1 Q2D IP or vehicle (PBS) control. Tumor volume (A and C) and body weight (B) were monitored for two weeks.

Figure 10. ADI-PEG 20 reduces CT26 tumor growth and prolongs survival as a single agent and in combination with anti-mPD-L1 mAb

CT26 cells were implanted into Balb/c mice and the next day the mice were treated with 12 mg/kg of ADI-PEG 20 BIWx5W IM, 10 mg/kg anti-mPD-L1 Q2D x 5W IP, 12 mg/kg of ADI-PEG 20 IM BIWx5W and 10 mg/kg anti-mPD-L1 IP Q2D x 5W or vehicle (PBS) control. Tumor Volume (A), body weight (B) and survival (C) were assessed over time.