Reversal of indoleamine 2,3-dioxygenase-mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme

Abstract

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8⁺ lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.

Figure 1. Kynureninase administration reduces tumor growth by depleting kynurenine from the tumor microenvironment

(a) Schematic of the proposed therapeutic mechanism of Kynureninase administration. (b) Mice were treated with vehicle control (PBS, n = 13) heat-deactivated control enzyme (20 mg/kg, n = 17) or PEG-Kynureninase (20 mg/kg, n = 37) four days after CT26 tumor implantation and administered every 72 hours thereafter for a total of 6 doses. Graph shows survival data compiled from three independent experiments. (c) Ten days after tumor challenge, mice with large, established B16-F10 tumors (104 mm³ mean +/− 9.5 mm³) were treated with PEG-Kynureninase (20 mg/kg) or heat-deactivated control enzyme (20 mg/kg) every 72 hours for a total of 6 doses. Graph shows survival data from three independent experiments (n = 14 mice per group). (d) Mice bearing large B16-F10 tumors (198 mm³ mean +/− 6.8 mm³) were treated with a single dose of PEG-Kynureninase (20 mg/kg) or heat-deactivated control enzyme. Serum and tumor samples were collected for LC-MS/MS metabolomics analysis at times indicated from three independent experiments, with each symbol representing data from individual mice and the red bars representing the mean +/− SEM (tumor analysis of all metabolites: n = 5 for deactivated enzyme control and n = 6 for PEG-Kynureninase treated groups at each time point; serum Kynurenine and Tryptophan: n = 13 per group at 4 hours, n = 14 mice per group at 24 hours, n = 4 mice per group at 48 hours and n = 5 mice per group at 72 hours; serum Anthranilic Acid: n = 9 mice per group at 4 hours and 24 hours, n = 4 mice per group at 48 hours and n = 3 mice per group at 72 hours). (e) Ido1^−/− (n = 10 per group) or (f) Rag^−/− (n = 8 per group) mice bearing established B16-F10 tumors (>89 mm³) were treated with PEG-Kynureninase (20 mg/kg) or heat-deactivated control enzyme (20 mg/kg) on day 10 and then every 72 hours thereafter for a total of 6 doses. Graphs show representative survival data from one of two independent experiments which had similar results. (g) Mice with established B16-F10 tumors were administered either CD8 depleting or isotype control antibodies and treated with PEG-Kynureninase (20 mg/kg) every 72 hours for a total of six doses staring on day 10. Graph depicts cumulative survival data compiled from two independent experiments with similar results (n = 13 mice per group). Statistical significance of treated mice compared to (b and c) control groups or (g) CD8 depleted mice was determined by Log-rank (Mantel-Cox) test.

Figure 2. Kynureninase treatment increases the frequency of effector CD8⁺ TIL

(a–e) Mice with large B16-F10 tumors (125 mm³ mean +/− 8 mm³, day 10) were treated with two doses of PEG-Kynureninase (20 mg/kg) or heat-deactivated control enzyme administered 72 hours apart. Tumors and spleens were analyzed 24 hours after the second treatment. CD8⁺ and CD4⁺ TIL in flow cytometric analysis were identified by surface expression of CD45 and T-cell markers. (a) Percent viable CD8⁺ TIL in tumor digests (n = 22 deactivated control and n = 24 PEG-Kynureninase compiled from seven independent experiments). (b) Shown are representative images of the margin and interior of frozen tumor sections along with insets from enlargements of the regions indicated by white dotted lines. Graph depicts the collective quantification of CD3⁺CD8⁺ cells from 10 fields view for both along the margin and interior of each tumor (n = 3 mice per group, data compiled from two independent experiments with similar results). Scale bars = 50 μm. (c and d) Representative flow cytometric plots of CD8⁺ TIL and splenic CD8⁺ T-cells evaluated for expression of (c) Ki67 (n = 13 per group, compiled from four independent experiments) and (d) Granzyme B (n = 16 deactivated control and n = 18 PEG-Kynureninase, compiled from six independent experiments). (e) Production of cytokines by CD8⁺ TIL after ex vivo stimulation (n = 9 deactivated control and n = 10 PEG-Kynureninase, compiled from three independent experiments). (c–e) Graphs depict cumulative tumor analysis with each symbol representing results from individual tumors and the red bars indicating the mean +/− SEM. Statistical significance was determined by unpaired t-test (two-tailed) for all analyses.

Figure 3. Combination of PEG-Kynureninase with other immunotherapies for the treatment of large established tumors in multiple tumor models

(a) Survival data for mice with large, established (117 mm³ mean +/− 6.5 mm³) B16-F10 tumors treated with vehicle control (n = 10), αPD-1 + deactivated enzyme (n = 21), or αPD-1 + PEG-Kynureninase (n = 21). Dosing schedule as shown (αPD-1: 10 mg/kg/dose, PEG-Kynureninase: 20 mg/kg/dose). Results are compiled from two independent experiments with similar results (n = 21 treated groups and n = 10 vehicle alone). (b) Survival data of mice bearing large (110 mm³ mean +/− 8 mm³) 4T1 tumors treated with vehicle control (n = 10), αCTLA4+deactivated enzyme (n = 12) or αCTLA4+PEG-Kynureninase (n = 12). Dosing schedule as shown (αCTLA4: 10 mg/kg/dose, PEG-Kynureninase: 20 mg/kg/dose). Graph depicts data from one experiment. (c) Mice challenged with 5 × 10⁵ CT26 cells were treated with Gp96-Ig vaccine (1 × 10⁶ CT26 cells transfected with Gp96-Ig), PEG-Kynureninase (20 mg/kg) or both as shown, with two doses of PEG-Kynureninase on day 10. Graph depicts representative data from one of two independent experiments with similar results (n = 4 untreated mice and n = 7 mice per treatment groups). Statistical significance of treatment groups in comparison to either control groups or between treatment groups (indicated by dashed lines) were determined by Log-rank (Mantel-Cox) test.