Reversal of tumoral immune resistance by inhibition of tryptophan 2,3-dioxygenase

Abstract

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO1) is an important mechanism of peripheral immune tolerance contributing to tumoral immune resistance, and IDO1 inhibition is an active area of drug development. Tryptophan 2,3-dioxygenase (TDO) is an unrelated hepatic enzyme that also degrades tryptophan along the kynurenine pathway. Here, we show that enzymatically active TDO is expressed in a significant proportion of human tumors. In a preclinical model, TDO expression by tumors prevented their rejection by immunized mice. We developed a TDO inhibitor, which, upon systemic treatment, restored the ability of mice to reject TDO-expressing tumors. Our results describe a mechanism of tumoral immune resistance based on TDO expression and establish proof-of-concept for the use of TDO inhibitors in cancer therapy.

Fig. 1.

Immune resistance of TDO-expressing tumors. (A) Mice (n = 45 per group) were immunized by i.p. injection of living L1210 cells expressing P1A and B7-1, and challenged 4 wk later by i.p. injection of 4 × 10⁵ cells either P815B cl1 (○), or P815B-mTDO cl8 (◇), or P815B-mTDO cl12 (●). Tumor progression was monitored. Shown are the compiled results of three experiments, each involving groups of 15 mice. P < 0.001 for cl1 versus cl8 and for cl1 versus cl12 (Logrank test). (B) Naïve (solid line) (n = 15 per group) or irradiated mice (dashed line) (n = 5 per group, 650 cGy) were injected as in A. One representative experiment out of five is shown. (C) Lysis of TDO-transfected P815B cells by P1A-specific CTLs. P1A-specific CTLs were incubated with ⁵¹Cr-loaded TDO-transfected P815B cells at varying effector/target ratios. P815 variant P1.istA-B-, which has lost gene P1A, was used as a control target (□) P1A-negative variant). One representative experiment out of three is shown. (D) Proportion of P1A-specific T cells among CD8+ T cells in the peritoneal cavity of immunized mice, estimated using H-2L^d/P1A tetramers 1 d before or 4 d after i.p. challenge with 10⁶ cells of P815B cl1 (○, n = 30 mice) or P815B-mTDO cl12 (●, n = 30 mice). Error bars represent SEM. P < 0.001 for cl1 versus cl12 after challenge (two-tailed Student t test). One representative experiment out of two is shown.

Fig. 2.

Reversal of immune resistance by systemic inhibition of TDO. (A) Structure, activity, physicochemical features, and bioavailability of TDO inhibitors 680C91 and LM10. K_i were measured on recombinant human TDO (hTDO) (16). A cellular assay based on mouse P815B-mTDO, mouse P815B-mIDO, or human 293E-hTDO cells was used to measure IC50, defined as the concentration giving 50% inhibition of TDO activity at a l-tryptophan concentration of 80 μM. Values ± SEM are shown corresponding to the mean of three independent experiments. LD50 was estimated by an MTT assay evaluating cell viability in the same cultures. Solubility was evaluated at room temperature as described in ref. . Bioavailability was estimated by measuring plasma concentration of the compounds after 1, 2, or 7 d of oral administration of 160 mg/kg/day. Mice (n = 10) received either normal drinking water or a solution of 1 mg/mL 680C91 at pH 2.5 or 1 mg/mL LM10 at pH 9, of which they drank an average of 4 mL/day. (B) Tumor progression in immunized mice (n = 45 per group) challenged by i.p. injection of 4 × 10⁵ cells from either P815B-mTDO cl8 (diamonds, Left), P815B-mTDO cl12 (triangles, Center), or P815B cl1 (circles, Right) and treated (black symbol) or not (white symbol) with 160 mg/kg/day LM10 in the drinking water, starting one day before the injection of tumor cells. Shown are the compiled results of three experiments, each involving groups of 15 mice. The untreated groups are identical to those shown on Fig. 1A. Mice received either normal drinking water or a solution of LM10 at 1 mg/mL P = 0.001, P < 0.001, and P = 0.006 for treated versus untreated mice after challenge with cl8, cl12 and cl1, respectively (Logrank test).

Fig. 3.

Lack of antitumor effect of LM10 in RAG2 knockout mice. RAG2 knockout mice (15 per group) received a s.c. injection of 2 × 10⁵ cells of the indicated P815 tumor clones (either P815B-mTDO cl8 (diamonds, Left), P815B-mTDO cl12 (triangles, Center), or P815B cl1 (circles, Right), and were treated (filled symbols) or not (open symbols) with 160 mg/kg/day LM10 in the drinking water (1 mg/mL) as in Fig. 2. The proportion of mice bearing progressive tumors in A and the mean tumor volume in B are reported. Tumor volume was calculated with formula (π x L x l²)/12. One representative experiment is shown out of two.