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.T ryptophan 2,3-dioxygenase (TDO) is a homotetrameric hemecontaining cytosolic enzyme encoded by gene TDO2 and expressed at high levels in the liver. It catalyses the first and ratelimiting step of tryptophan degradation along the kynurenine pathway and thereby regulates systemic tryptophan levels. The same reaction can be catalyzed by another heme-containing cytosolic enzyme, named indoleamine 2,3-dioxygenase (IDO1), which has no sequence similarity with TDO, is not expressed in the liver, and is monomeric. IDO1 has been the focus of attention in recent years because of its immunosuppressive effects on T lymphocytes, resulting partly from tryptophan depletion and partly from direct effects of tryptophan catabolites (1-3). IDO1 is expressed constitutively in the placenta, where it plays a key role in feto-maternal tolerance (2), and in many tumors, where it contributes to tumoral resistance to immune rejection (4-7). IDO1 expression is also inducible in many cells, including dendritic cells, and appears to play a role in peripheral immune tolerance and the retro-control of immune responses (8). In contrast, little is known about the effect of TDO expression on the immune response. A recent report indicated that human cells transfected with TDO depleted tryptophan and thereby prevented both the growth of pathogens and the proliferation of allogeneic T lymphocytes (9). These results suggested that TDO might mediate immunosuppressive effects similar to those of IDO1. We set out to examine whether tumor cells express TDO and thereby inhibit T-cell-mediated immune responses. ResultsWe first observed that many human tumor samples expressed gene TDO2 as measured by real-time RT-PCR (Table 1). This was the case for 41% of bladder carcinomas, 50% of melanomas and 100% of hepatocarcinomas. To confirm the activity of TDO in tumor cells, we selected a series of human tumor cell lines that also expressed the TDO2 mRNA. We incubated cells for 24 h in medium containing a known concentration of tryptophan, and measured by HPLC in the supernatant the concentration of tryptophan and kynurenine, which is the main tryptophan catabolite (Table 2). HEK-293 cells transfected or not with human TDO2 were used as pos...

Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tumoral resistance to immune rejection. In humans, constitutive expression of IDO1 has been observed in several tumor types. However, a comprehensive analysis of its expression in normal and tumor tissues is still required to anticipate the risks and potential benefits of IDO1 inhibitors. Using a newly validated monoclonal antibody to human IDO1, we performed an extensive immunohistochemical analysis of IDO1 expression in normal and tumor tissues. In normal tissues, IDO1 was expressed by endothelial cells in the placenta and lung and by epithelial cells in the female genital tract. In lymphoid tissues, IDO1 was expressed in mature dendritic cells with a phenotype (CD83 þ ,

Indoleamine 2,3-dioxygenase (IDO) is an important therapeutic target for the treatment of diseases such as cancer that involve pathological immune escape. We have used the evolutionary docking algorithm EADock to design new inhibitors of this enzyme. First, we investigated the modes of binding of all known IDO inhibitors. On the basis of the observed docked conformations, we developed a pharmacophore model, which was then used to devise new compounds to be tested for IDO inhibition. We also used a fragment-based approach to design and to optimize small organic molecule inhibitors. Both approaches yielded several new low-molecular weight inhibitor scaffolds, the most active being of nanomolar potency in an enzymatic assay. Cellular assays confirmed the potential biological relevance of four different scaffolds.

Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the MHC and recognized by CTLs. In this study, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two noncontiguous fragments of the melanoma differentiation Ag gp100PMEL17 spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3–6 aa, the peptide described in this work results from the ultimate association of an 8-aa fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least 3 aa long to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C terminus that needs to be further trimmed to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described in this work.

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO) is an important mechanism of peripheral immune tolerance contributing to tumoral immune resistance. IDO inhibition is thus an active area of research in drug development. Recently, our group has shown that tryptophan 2,3-dioxygenase (TDO), an unrelated hepatic enzyme also catalyzing the first step of tryptophan degradation, is also expressed in many tumors and that this expression prevents tumor rejection by locally depleting tryptophan. Herein, we report a structure-activity study on a series of 3-(2-(pyridyl)ethenyl)indoles. More than 70 novel derivatives were synthesized, and their TDO inhibitory potency was evaluated. The rationalization of the structure-activity relationships (SARs) revealed essential features to attain high TDO inhibition and notably a dense H-bond network mainly involving His(55) and Thr(254) residues. Our study led to the identification of a very promising compound (58) displaying good TDO inhibition (K(i) = 5.5 μM), high selectivity, and good oral bioavailability. Indeed, 58 was chosen for preclinical evaluation.

a b s t r a c tIndoleamine 2,3-dioxygenase (IDO) is an important new therapeutic target for the treatment of cancer. With the aim of discovering novel IDO inhibitors, a virtual screen was undertaken and led to the discovery of the keto-indole derivative 1a endowed with an inhibitory potency in the micromolar range. Detailed kinetics were performed and revealed an uncompetitive inhibition profile. Preliminary SARs were drawn in this series and corroborated the putative binding orientation as suggested by docking.

The uptake and long-term cross-presentation of tumor Ag long peptides (LP) by dendritic cells (DC) make them attractive cancer vaccine candidates. However, it remains to be established whether LP can prime long-lived tumor-reactive CTL and whether other cell types are able to cross-present them. Using HLA-A2 healthy donor and melanoma patient-derived PBMC, we studied the in vitro cross-priming potential of Melan-A 16–40 LP bearing the HLA-A2–restricted epitope 26–35 or its analog 26–35A27L and compared it to the priming capacity of the short analog. We then addressed LP priming capacity in vivo using HLA-A2 mice. We also studied LP cross-presentation by monocyte-derived DC, plasmacytoid DC, monocytes, and B cells. We showed that the modified LP gave rise to high and sustained cross-presentation by monocyte-derived DC. This led to cross priming in vitro and in vivo and to expansion of long-lived tumor-reactive cytotoxic T cells. In contrast, the LP containing the natural 26–35 epitope primed specific T cells poorly, despite its long-lived cross-presentation, and T cells primed against the short analog were short-lived. We further showed that LP cross-presentation is restricted to monocytes and conventional DC. These results document for the first time, to our knowledge, the strong immunogenicity of a human tumor Ag LP. Of note, they underscore that this property is critically dependent on sufficient HLA binding affinity and/or TCR ligand potency of the cross-presented epitope. We conclude that LP fulfilling this requirement should be used as tumor vaccines, together with DC maturating agents, especially the Melan-A 16–40A27L LP, for the treatment of HLA-A2+ melanoma patients.

The use of dendritic cells (DC) for the development of therapeutic cancer vaccines is attractive because of their unique ability to present tumor epitopes via the MHC class I pathway to induce cytotoxic CD8+ T lymphocyte responses. C-Type membrane lectins, DC-SIGN and the mannose receptor (MR), present on the DC surface, recognize oligosaccharides containing mannose and/or fucose and mediate sugar-specific endocytosis of synthetic oligolysine-based glycoclusters. We therefore asked whether a glycotargeting approach could be used to induce uptake and presentation of tumor antigens by DC. To this end, we designed and synthesized glycocluster conjugates containing a CD8+ epitope of the Melan-A/Mart-1 melanoma antigen. These glycocluster-Melan-A conjugates were obtained by coupling glycosynthons: oligosaccharyl-pyroglutamyl-beta-alanine derivatives containing either disaccharides, a dimannoside (Manalpha-6Man) or lactoside, or a Lewis oligosaccharide, to Melan-A 16-40 peptide comprising the 26-35 HLA-A2 restricted T cell epitope, extended with an oligolysine stretch at the C-terminal end. We showed by confocal microscopy and flow cytometry that fluorescent-labeled Melan-A glycoclusters containing either dimannoside or Lewis oligosaccharide were taken up by DC and concentrated in acidic vesicles; conversely lactoside glycopeptides were not at all taken up. Furthermore, using surface plasmon resonance, we showed that dimannoside and Lewis-Melan-A conjugates bind MR and DC-SIGN with high affinity. DC loaded with these conjugates, but not with the lactose-Melan-A conjugate, led to an efficient presentation of the Melan-A epitope eliciting a CD8+ T-lymphocyte response. These data suggest that synthetically designed glycocluster-tumor antigen conjugates may induce antigen cross-presentation by DC and represent a promising tool for the development of tumor vaccines.