Tryptophan-catabolizing Enzyme Research Articles

IDO1 Deficiency Does Not Affect Disease in Mouse Models of Systemic Juvenile Idiopathic Arthritis and Secondary Hemophagocytic Lymphohistiocytosis.

ObjectivesIndoleamine 2,3-dioxygenase-1 (IDO1) is an immune-modulatory enzyme that catalyzes the degradation of tryptophan (Trp) to kynurenine (Kyn) and is strongly induced by interferon (IFN)-γ. We previously reported highly increased levels of IFN-γ and corresponding IDO activity in patients with hemophagocytic lymphohistiocytosis (HLH), a hyper-inflammatory syndrome. On the other hand, IFN-γ and IDO were low in patients with systemic juvenile idiopathic arthritis (sJIA), an autoinflammatory syndrome. As HLH can occur as a complication of sJIA, the opposing levels of both IFN-γ and IDO are remarkable. In animal models for sJIA and HLH, the role of IFN-γ differs from being protective to pathogenic. In this study, we aimed to unravel the role of IDO1 in the pathogenesis of sJIA and HLH.MethodsWild-type and IDO1-knockout (IDO1-KO) mice were used in 3 models of sJIA or HLH: complete Freund’s adjuvant (CFA)-injected mice developed an sJIA-like syndrome and secondary HLH (sHLH) was evoked by either repeated injection of unmethylated CpG oligonucleotide or by primary infection with mouse cytomegalovirus (MCMV). An anti-CD3-induced cytokine release syndrome was used as a non-sJIA/HLH control model.ResultsNo differences were found in clinical, laboratory and hematological features of sJIA/HLH between wild-type and IDO1-KO mice. As IDO modulates the immune response via induction of regulatory T cells and inhibition of T cell proliferation, we investigated both features in a T cell-triggered cytokine release syndrome. Again, no differences were observed in serum cytokine levels, percentages of regulatory T cells, nor of proliferating or apoptotic thymocytes and lymph node cells.ConclusionsOur data demonstrate that IDO1 deficiency does not affect inflammation in sJIA, sHLH and a T cell-triggered cytokine release model. We hypothesize that other tryptophan-catabolizing enzymes like IDO2 and tryptophan 2,3-dioxygenase (TDO) might compensate for the lack of IDO1.

Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway

A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.

Abstract P2-11-09: A phase 2 randomized trial of the IDO pathway inhibitor indoximod in combination with taxane based chemotherapy for metastatic breast cancer: Preliminary data

Abstract Background: Indoleamine 2,3 dioxygenase (IDO) is a tryptophan-catabolizing enzyme that plays a key role in the normal regulation of peripheral immune tolerance. Tumors also employ this mechanism to induce a state of immunosuppression, evading immune mediated destruction. Indoximod (D-1-methyltryptophan) is a broad IDO pathway inhibitor as it has been shown to potentially interfere with multiple targets within the IDO pathway. Preclinical studies in MMTV-neu mouse models have demonstrated that combining indoximod with cytotoxic chemotherapy had a greater in-vivo anti-tumor effect than either agent alone. A phase 1 trial combining docetaxel and indoximod demonstrated safety and responses in metastatic breast cancer (mBC) patients. Based on these data a phase 2 trial evaluating indoximod in combination with taxane chemotherapy as first line therapy in patients with metastatic breast cancer was initiated. Methods: The study is a 1:1 randomized, placebo controlled, four arm phase 2 trial. The study treatment is docetaxel 75mg/m2 IV D8 plus indoximod 1200mg PO BID D1-14 every 21 days or matching placebo in study arms 1A and 2A or paclitaxel 80 mg/m2 IV weekly 3 out of 4 weeks with indoximod or matching placebo given BID Days 1-14 in arms 1B and 2B. The primary endpoint is progression free survival. Secondary endpoints include overall survival, response rate, safety, and immune response correlative assays. Patients with measurable, histologically confirmed mBC, no prior chemotherapies (hormonal therapies allowed) in the metastatic setting, ER+ or ER –, HER2 -, ECOG PS 0-1, no active CNS disease, no active autoimmune disease are eligible. Target enrollment is 154 patients. Results: At submission, 65 patients have evaluable safety data on trial. The pooled, blinded data was evaluated for any safety signals. Patient demographics include median age 58 years, 95% female, 17% African American, 25% triple-negative disease, and 82% with multiple sites of metastatic disease. 28% of patients had at least one Grade 3 or 4 adverse event. Those occurring in multiple patients include dyspnea 5%, pleural effusion 3%, non-cardiac chest pain 3%, and febrile neutropenia 3%. Most patients had at least one adverse event (86%). The most common were anemia 14%, tearing 11%, constipation 19%, diarrhea34%, nausea 44%, vomiting 22%, fatigue 48%, peripheral edema 17%, increased liver function tests 10%, lymphopenia 17%, hyperglycemia 15%, myalgia 14%, dizziness 14%, dysguesia 16%, headache 20%, peripheral neuropathy 12%, dyspnea 19%, alopecia 34%, and rash 14%, all compatible with the side effect profile of single agent taxanes. No immune specific serious advents were reported. No treatment related deaths have been reported. Conclusion: The aggregate safety data for these 65 patient are similar to what is typically observed with docetaxel and paclitaxel. No unexpected serious immune linked adverse events were reported. The trial is currently open at multiple clinical sites in the US and Europe and actively enrolling patients. Updated data will be presented. NCT01191216. Citation Format: Tang S-C, Montero A, Munn D, Link C, Vahanian N, Kennedy E, Soliman H. A phase 2 randomized trial of the IDO pathway inhibitor indoximod in combination with taxane based chemotherapy for metastatic breast cancer: Preliminary data. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-11-09.

Local Generation of Kynurenines Mediates Inhibition of Neutrophil Chemotaxis by Uropathogenic Escherichia coli.

During epithelial infections, pathogenic bacteria employ an array of strategies to attenuate and evade host immune responses, including the influx of polymorphonuclear leukocytes (PMN; neutrophils). Among the most common bacterial infections in humans are those of the urinary tract, caused chiefly by uropathogenic Escherichia coli (UPEC). During the establishment of bacterial cystitis, UPEC suppresses innate responses via multiple independent strategies. We recently described UPEC attenuation of PMN trafficking to the urinary bladder through pathogen-specific local induction of indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolic enzyme previously shown to have regulatory activity only in adaptive immunity. Here, we investigated the mechanism by which IDO induction attenuates PMN migration. Local tryptophan limitation, by which IDO is known to influence T cell longevity and proliferation, was not involved in its effect on PMN trafficking. Instead, metabolites in the IDO pathway, particularly L-kynurenine, directly suppressed PMN transepithelial migration and induced an attached, spread morphology in PMN both at rest and in the presence of chemotactic stimuli. Finally, kynurenines represent known ligands of the mammalian aryl hydrocarbon receptor (AHR), and UPEC infection of Ahr(-/-)mice recapitulated the derepressed PMN recruitment observed previously in Ido1(-/-)mice. UPEC therefore suppresses neutrophil migration early in bacterial cystitis by eliciting an IDO-mediated increase in local production of kynurenines, which act through the AHR to impair neutrophil chemotaxis.

Results of the phase Ib portion of a phase I/II trial of the indoleamine 2,3-dioxygenase pathway (IDO) inhibitor indoximod plus gemcitabine/nab-paclitaxel for the treatment of metastatic pancreatic cancer.

452 Background: IDO is a tryptophan-catabolizing enzyme that plays a key role in the normal regulation of peripheral immune tolerance. Tumors also employ this mechanism to induce a state of immunosuppression. In cancer, IDO mediates an acquired immune tolerance towards tumors, allowing evasion of immune mediated destruction. Indoximod is a broad IDO pathway inhibitor, as it has been shown to potentially interfere with multiple targets within the IDO pathway. Pre-clinical models have demonstrated synergy between indoximod and chemotherapy. The combination of gemcitabine (Gem) and nab-paclitaxel (Nab) is a current SOC for first line treatment of metastatic pancreas cancer. This trial is designed to determine the potential for benefit of combination therapy with indoximod and Gem / Nab for to patients with metastatic pancreatic cancer. Methods: Indoximod was escalated (600mg/1000mg/1200mg PO twice daily continuous dosing) in combination with Gem / Nab (1000mg/m2 / 125mg/m2 q week x 3 per 4 week cycle) in a 3+3 design. Patients were first line metastatic pancreatic cancer or minimum 6 months from adjuvant chemo and / or radiation following previous resection. Treatment continues until progression or toxicity. Primary endpoints for Phase 1 include safety, toxicity, and determination of a Phase 2 dose. The prospective collection of tumor samples for exploration of biomarkers is built into the trial. Results: 15 patients were required to successfully dose escalate the Phase 1 study to 1200 mg twice daily. Two patients were replaced in the lowest dose cohort after rapid deterioration due to underlying disease during the regimen limiting toxicity (RLT) window. One RLT was observed during the study (ascites Grade 3) at the highest dose cohort. The most common AE’s (all Grade 1 or 2) occurring in ≥ 4 subjects, regardless of attribution, were nausea, fatigue, peripheral edema, peripheral neuropathy, alopecia. Conclusions: Indoximod and Gem / Nab were well tolerated in a clinical trial setting. The Phase 2 dose was set at 1200 mg twice daily and Phase 2 enrollment (target 80 patients) is ongoing. Updated results will be presented. Clinical trial information: NCT02077881.

Abstract A16: The crosstalk between Indoleamine 2,3 Dioxygenase (IDO) and cancer stem cells in murine melanoma: A potential modality of local control

Abstract Introduction: Indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of tryptophan catabolism, has been strongly associated with the progression of malignancy and poor survival in melanoma patients. As a result, IDO is a leading target for interventions aimed at restoring melanoma immune surveillance. IDO, which is produced by cells including dendritic cells, macrophages, stem cells, and most tumor cells, potently suppresses immune responses by depleting local tryptophan and allowing the accumulation of tryptophan metabolites. However, the actual cellular and molecular mechanisms underlying the immunoregulatory function of IDO remain largely unknown. The relatively newly described cancer stem cells (CSCs) are tumor-initiating cells (TICs) that often survive therapy and give rise to second-line tumors by self-renewal and differentiation into multiple cell types. CSCs are proposed to persist in tumors as a distinct population and to cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially those with metastatic disease. Methods: C57/BL6 mice were injected with B16F10 murine melanoma cell line (1 x 106 cells/mouse, subcutaneous). Tumor growth monitored. At 3mm, tumors were injected with an IDO Inhibitor (1 MT) (Group 1) twice weekly (20 mg/mouse per injection). Controls (Group 2) received vehicle only (PBS), Group 3 weekly Cisplatin (5mg/kg), and Group 4 both Cisplatin and 1MT (IDO inhibitor). Once the first tumor reached 12mm in size, the mice were sacrificed and tumor cells subjected to flow-cytometry, and statistical analysis, and p-values <0.05 considered significant. Results: Our data showed for the first time that IDO was expressed by CSCs in a murine melanoma model. These IDO-expressing CSCs may be capable of suppressing T lymphocyte infiltration into the tumor microenvironment, which was reversed by the IDO inhibitor, 1-methyl-tryptophan. Conclusion: Our findings offer an important new line of evidence that interventional targeting of IDO activity could be used to restore tumor immunity by relieving IDO-mediated immune suppression of CSCs in the tumor microenvironment as well as in tumor cells themselves. Citation Format: Marsha Kocherla, Blake Christianson, James Walker, Xu Qin, Jack C. Yu, Babak Baban. The crosstalk between Indoleamine 2,3 Dioxygenase (IDO) and cancer stem cells in murine melanoma: A potential modality of local control. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A16.

The Role of Indoleamine 2,3-Dioxygenase in Diethylnitrosamine-Induced Liver Carcinogenesis.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing intracellular enzyme of the L-kynurenine pathway, causes preneoplastic cells and tumor cells to escape the immune system by inducing immune tolerance; this mechanism might be associated with the development and progression of human malignancies. In the present study, we investigated the role of IDO in diethylnitrosamine (DEN)-induced hepatocarcinogenesis by using IDO-knockout (KO) mice. To induce hepatocellular carcinoma (HCC), hepatic adenoma, and preneoplastic hepatocellular lesions termed foci of cellular alteration (FCA), male IDO-wild-type (WT) and IDO-KO mice with a C57BL/6J background received a single intraperitoneal injection of DEN at 2 weeks of age. The mice were sacrificed to evaluate the development of FCA and hepatocellular neoplasms. HCC overexpressed IDO and L-kynurenine compared to surrounding normal tissue in the DEN-treated IDO-WT mice. The number and cell proliferative activity of FCAs, and the incidence and multiplicity of HCC were significantly greater in the IDO-WT than in the IDO-KO mice. The expression levels of the IDO protein, of L-kynurenine, and of IFN-γ, COX-2, TNF-α, and Foxp3 mRNA were also significantly increased in the DEN-induced hepatic tumors that developed in the IDO-WT mice. The mRNA expression levels of CD8, perforin and granzyme B were markedly increased in hepatic tumors developed in IDO-KO mice. Moreover, Foxp3-positive inflammatory cells had infiltrated into the livers of DEN-treated IDO-WT mice, whereas fewer cells had infiltrated into the livers of IDO-KO mice. Induction of IDO and elevation of L-kynurenine might play a critical role in both the early and late phase of liver carcinogenesis. Our findings suggest that inhibition of IDO might offer a promising strategy for the prevention of liver cancer.

UV-B Exposure Increases the Activity of Indoleamine 2, 3-Dioxygenase (Ido) and Alters the Levels of Tryptophan Metabolites in Indian Ground Squirrel (Funambulus Palmarum) Lens

The ocular lens of the ground squirrel is an excellent model for studies of eye light interactions that may apply to the human system. UV-B radiation induced damage to the eye lens is believed to be mediated through ROS Antioxidant systems, both enzymatic and non- enzymatic aid in preventing the damage to the lens. The activity of indoleamine 2,3- dioxygenase (IDO), a first rate limiting enzyme of tryptophan catabolism, has been studied in the lenses of Indian ground squirrel exposed to UV-B radiation. Further, antioxidants and oxidative stress markers have also been investigated to assess the oxidative status. Our investigation revealed an increased indoleamine 2,3-dioxygenase activity and tryptophan metabolites suggesting their protective role in UV stress. The study indicates that UV-B irradiation for 12 h and 24 h (300 nm; 100 μw/cm2) of lens samples led to a marginal decrease in GSH and increase in carbonyls and MDA levels reflecting the role of indoleamine 2,3-dioxygenase in protecting the lens tissue.

Cell Intrinsic Factors Modulate the Effects of IFNγ on the Development of Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that cannot synthesize several amino acids, including tryptophan. Rather, C. trachomatis acquires these essential metabolites from its human host cell. Chlamydial dependence on host-provided tryptophan underlies a major host defense mechanism against the bacterium; namely, the induction of the host tryptophan-catabolizing enzyme, indoleamine 2,3- dioxygenase (IDO1) by interferon gamma (IFNγ), which leads to eradication of C. trachomatis by tryptophan starvation. For this reason, IFNγ is proposed to be the major host protective cytokine against genital C. trachomatis infections. The protective effect of IFNγ against C. trachomatis can be recapitulated in vitro using epithelial cell-lines such as the cervical carcinoma derived cell-line Hela, the Hela subclone HEp-2, and the cervical carcinoma derived cell-line ME180. Addition of IFNγ to these cells infected with C. trachomatis results in a strong bactericidal or bacteriostatic effect dependent on the concentration of IFNγ administered. Unlike Hela, HEp-2, and ME180, there are other human epithelial, or epithelial-like cell-lines where administration of IFNγ does not affect chlamydial replication, although they express the IFNγ receptor (IFNGR). In this report, we have characterized the mechanisms that underlie this dichotomy using the cell-lines C33A and 293. Akin to Hela, C33A is derived from a human cervical carcinoma, while 293 cells were produced by transfection of adenovirus type 5 DNA into embryonic kidney cells. We demonstrate that although IFNGR is expressed at high levels in C33A cells, its ligation by IFNγ does not result in STAT1 phosphorylation, an essential step for activation of the IDO1 promoter. Our results indicate that although the IFNγ-dependent signaling cascade is intact in 293 cells; the IDO1 promoter is not activated in these cells because it is epigenetically silenced, most likely by DNA methylation. Because polymorphisms in IFNγ, IFNGR, and the IDO1 promoter are known to affect other human infections or diseased states, our results indicate that the effect of allelic differences in these genes and the pathways they activate should be evaluated for their effect on C. trachomatis pathology.

Preliminary results from a Phase I/II study of epacadostat (incb024360) in combination with pembrolizumab in patients with selected advanced cancers

Meeting abstracts Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolizing enzyme that is expressed in many cancers and induces immune tolerance by suppressing T cell responses. Epacadostat is a potent, selective oral inhibitor of IDO1. A dose-escalation study of epacadostat with

IMPS-43IDO1 DOES NOT FUNCTION AS A TRYPTOPHAN CATABOLIC ENZYME IN MALIGNANT GLIOMA

The upregulation of indoleamine 2,3 dioxygenase 1 (IDO1) in glioblastoma (GBM) is associated with significantly decreased survival in patients. We have previously demonstrated that glioma-derived IDO1 is required for Treg accumulation and immune-evasion of brain tumors in the immunocompetent intracranial GL261 model of GBM. Notably, IDO1-deficient (IDO1-/-) mice bearing GL261 brain tumors have no deficit in tumor-infiltrating Treg levels and an overall survival similar to wild-type (WT; IDO1-competent) mice. Since IDO1 is associated with the catabolic conversion of tryptophan (Trp) into kynurenine (Kyn), combined with recent clinical efforts aimed at inhibiting IDO1, we sought to determine the enzymatic contribution of IDO1 in brain tumors. WT and IDO1-/- mice intracranially-injected IDO1-competent or -deficient GL261 cells were analyzed by HPLC to determine total Trp and Kyn levels in the serum and brain. Indicative of enzymatic activity, naive (non-tumor bearing) WT mice possessed a Kyn/Trp ratio in the serum and brain of 2.44 ± 0.09 and 2.22 ± 0.32, respectively, which was significantly higher than in IDO1-/- mice (0.374 ± 0.09 and 0.834 ± 0.05, respectively) (P < 0.01; n = 5-6/group). Unexpectedly, WT mice bearing 2 week post-intracranially-injected (2wp-ic.) IDO1-competent and -deficient GL261 tumors possessed a brain Kyn/Trp ratio of 2.04 ± 0.05 and 1.88 ± 0.17, respectively, which was not significantly different from one another. In contrast, IDO1-/- mice bearing 2wp-ic. IDO1-competent and -deficient tumors possessed a brain Kyn/Trp ratio of 0.77 ± 0.06 and 0.72 ± 0.03, respectively, which was not different from one another, but significantly decreased when compared to respective tumors from WT mice (P < 0.01; n = 4-7/group). To the best of our knowledge, these novel findings suggest for the first time that, IDO1 does not behave as a tryptophan catabolic enzyme in brain tumor cells. Given the indispensable role of IDO1 in mediating immune-evasion to GBM, we are currently pursuing this mechanism.

The tryptophan metabolite picolinic acid suppresses proliferation and metabolic activity of CD4+ T cells and inhibits c-Myc activation.

Tryptophan metabolites, including kynurenine, 3-hydroxyanthranilic acid, and picolinic acid, are key mediators of immunosuppression by cells expressing the tryptophan-catabolizing enzyme indoleamine2,3-dioxygenase. In this study, we assessed the influence of picolinic acid on cell viability and effector functions of CD4(+)T cells following in vitro activation with agonistic anti-CD3/anti-CD28 antibodies. In contrast to kynurenine and 3-hydroxyanthranilic acid, exposure of T cells with picolinic acid did not affect cell viability, whereas proliferation and metabolic activity were suppressed in a dose-dependent manner. On the other hand, cytokine secretion and up-regulation of cell surface activation markers were not or only weakly inhibited by picolinic acid. Picolinic acid exposure induced a state of deep anergy that could not be overcome by the addition of exogenous IL-2 and inhibited Th cell polarization. On the molecular level, important upstream signaling molecules, such as the MAPKs ERK and p38 and the mammalian target of rapamycin target protein S6 ribosomal protein, were not affected by picolinic acid. Likewise, NFAT, NF-κB, and AP-1 promoter activity in Jurkat T cells was not influenced by exposure to picolinic acid. Whereas transcriptional levels of v-myc avian myelocytomatosis viral oncogene homolog were not affected by picolinic acid, phosphorylation at Ser62 was strongly reduced in picolinic acid-exposed T cells following activation. In conclusion, picolinic acid mediates a unique immunosuppressive program in T cells, mainly inhibiting cell cycle and metabolic activity, while leaving other effector functions intact. These functional features are accompanied by reduced phosphorylation of v-myc avian myelocytomatosis viral oncogene homolog. It remains to be determined whether this effect is mediated by direct inhibition of ERK activity or whether indirect mechanisms apply.

Learning from other diseases: protection and pathology in chronic fungal infections.

Fungal commensals coexist in a complex milieu of bacteria within the human body. An increased understanding of the importance of microbiota in shaping the host's immune and metabolic activities has rendered fungal interactions with their hosts more complex than previously appreciated. Metagenomics has revealed the complex interactions between fungal and bacterial commensals that, either directly or through the participation of the host immune system, impact on immune homeostasis at mucosal surfaces that, in turn, lead to secondary fungal infections. Metabolomics has captured the dialogue between the mammalian host and its microbiota. It appears that the host tryptophan catabolic enzyme, indoleamine 2,3-dioxygenase 1 (IDO1) plays a dominant role in the interplay between tryptophan catabolism by microbial communities, the host's own pathway of metabolite production, and the activation of the aryl hydrocarbon receptor (AhR)/IL-22 axis, eventually impacting on mucosal immune homeostasis and host/fungal symbiosis. Thus, the regulatory loop involving AhR and IDO1 may be exploited for the development of multi-pronged host- and microbiota-directed therapeutic approaches for mucosal and systemic fungal diseases.

B Cell-Intrinsic IDO1 Regulates Humoral Immunity to T Cell-Independent Antigens.

Humoral responses to nonproteinaceous Ags (i.e., T cell independent [TI]) are a key component of the early response to bacterial and viral infection and a critical driver of systemic autoimmunity. However, mechanisms that regulate TI humoral immunity are poorly defined. In this study, we report that B cell-intrinsic induction of the tryptophan-catabolizing enzyme IDO1 is a key mechanism limiting TI Ab responses. When Ido1(-/-) mice were immunized with TI Ags, there was a significant increase in Ab titers and formation of extrafollicular Ab-secreting cells compared with controls. This effect was specific to TI Ags, as Ido1 disruption did not affect Ig production after immunization with protein Ags. The effect of IDO1 abrogation was confined to the B cell compartment, as adoptive transfer of Ido1(-/-) B cells to B cell-deficient mice was sufficient to replicate increased TI responses observed in Ido1(-/-) mice. Moreover, in vitro activation with TLR ligands or BCR crosslinking rapidly induced Ido1 expression and activity in purified B cells, and Ido1(-/-) B cells displayed enhanced proliferation and cell survival associated with increased Ig and cytokine production compared with wild-type B cells. Thus, our results demonstrate a novel, B cell-intrinsic, role for IDO1 as a regulator of humoral immunity that has implications for both vaccine design and prevention of autoimmunity.

Abstract 4469: A novel class of inhibitors of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) with potential for the treatment of cancer

Abstract A broad range of cancers express the tryptophan catabolising enzyme indoleamine 2,3-dioxygenase 1 (IDO1) as a mechanism of suppressing the endogenous anti-tumour immunity. High IDO1 expression by their tumours is associated with poor patient prognosis and has prompted the discovery of small molecule inhibitors of the enzyme for development as anti-cancer agents. Towards this end, we carried out a high throughput screen of 42,000 small molecules in the Walter and Eliza Hall Institute for Medical Research compound library and retrieved over 30 good hits with diverse structures that inhibited recombinant human IDO1 with potencies in the low μM range. Initial structure activity relationship studies of the best hits led us to focus on one chemical class, which, although less potent than some of the other hits on the list, exhibited a number of features that make it more favourable for development as a therapeutic agent. SN35837, the non-optimised hit from the high throughput screen is highly cell permeable and displays improved potency in inhibiting cytoplasmic hIDO1 in cell-based assays, compared to that for enzymatic assays. Complete inhibition of IDO1 function is seen at concentrations that has no measurable effects on cell viability. The imidazole lead from New Links Genetics, NLG919 has decreased activity in cells, whilst Incyte's hydroxyamidine inhibitor INCB024360 has similar potency in cellular and enzymatic assays. The maximum tolerated dose SN35837 in C57Bl/6 mice given as a single intraperitoneal bolus dose was 500 mg/kg. Mice tolerated daily intraperitoneal or subcutaneous administrations of SN35837 at 150 mg/kg. Statistically significant inhibition of growth of subcutaneously implanted GL261 gliomas and Lewis Lung carcinomas in syngeneic hosts were achieved after daily administration of 75 mg/kg SN35837. Preliminary studies indicate that the non-optimised hit also synergises when given in combination with anti-PD1 and anti-CTLA-4 antibodies. We conclude that derivatives of SN35837 may provide effective and potent inhibitors of IDO1 suitable for development as anti-cancer agents. Citation Format: Lai-Ming Ching, Brian D. Palmer, Petr Tomek, Jack U. Flanagan, Kimiora Henare. A novel class of inhibitors of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) with potential for the treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4469. doi:10.1158/1538-7445.AM2015-4469

Abstract 5223: A novel pro-angiogenic role for IDO1 in inflammatory tumor promotion

Abstract Chronic inflammation is a major contributing factor in cancer, but, due to the complex multifactorial nature of inflammation, there remains limited understanding of specific pathogenic determinants that might be targeted therapeutically. The tryptophan-catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase) has emerged as an intriguing, pro-tumorigenic regulator of immune function in this regard. Because IDO1 can be elevated in chronic inflammatory states even prior to the initiation of cancer, it may represent one of the earliest determinants directing the immune response towards supporting rather than eliminating tumors. Genetic studies in mice have clearly established the tumor-promoting role of IDO1, but what this actually entails remains uncertain. Here we present evidence to support the novel hypothesis that a principle means by which IDO1 facilitates tumorigenesis is by mitigating immune-based angiostasis. Tumor angiogenesis is characterized by excessive and disorganized blood vessel growth much like that induced by ischemia where immune cells have been shown to be important for limiting neovascularization. Likewise, an anti-angiogenic response may be a factor in tumor immunity. In particular, IFNγ, an inflammatory cytokine, long recognized as a major inducer of IDO1, has been shown to exert angiostatic activity against developing tumors, which was implicated in these studies as the primary mechanism for both CD4 and CD8 T cell dependent tumor rejection. Our recently reported finding that the loss of IDO1 resulted in diminished pulmonary vascularization (Smith, Cancer Discovery 2012) suggested the possibility that IDO1 might be working at cross purposes to limit IFNγ-mediated angiostasis. In this same study IDO1 loss was also associated with the attenuated induction of the pro-angiogenic inflammatory cytokine IL6. To directly investigate the role of IDO1 in pathologic angiogenesis, we have utilized an oxygen-induced retinopathy (OIR) model. As predicted, neovascularization in the OIR model was significantly reduced in Ido1-/- mice. Consistent with the hypothesis that IDO1 supports neovascularization primarily by counteracting the angiostatic activity of IFNγ, neovascularization in double knockout Ifng-/- Ido1-/- mice reverted back to wild type levels. Il6-/- mice, on the other hand, exhibited reduced neovascularization which was likewise reversed by the concurrent elimination of IFNγ. In conjunction with these angiogenesis studies, we have also examined pulmonary metastasis development by 4T1 breast carcinoma isografts. Loss of either IDO1 or IL6 resulted in resistance to pulmonary metastases that, in both cases, was abrogated by the concurrent loss of IFNγ. Taken together, these findings have led us to propose a conceptually novel working hypothesis that, in the context of an inflammatory cytokine milieu, IDO1 plays a key role in supporting tumor angiogenesis. Citation Format: Arpita Mondal, James B. DuHadaway, Erika Sutanto-Ward, Courtney Smith, George C. Prendergast, Arturo Bravo-Nuevo, Alexander J. Muller. A novel pro-angiogenic role for IDO1 in inflammatory tumor promotion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5223. doi:10.1158/1538-7445.AM2015-5223

Indolamine 2,3-dioxygenase expression by monocytes and dendritic cell populations in hepatitis C patients.

Dendritic cells (DCs) play an important role in the induction of the primary immune response to infection. DCs may express the tryptophan-catabolizing enzyme indolamine2,3-dioxygenase (IDO), which is an inducer of immune tolerance. Because there is evidence that chronic hepatitis C virus (HCV) infection leads to functional impairment of certain DC populations, we analysed IDO expression in DCs and monocytes from chronically infected and recovered HCV patients. The IDO1 and -2 expression was increased significantly in the monocytes of chronic HCV patients but, interestingly, not in those from recovered patients. The myeloid DCs from chronically infected HCV patients also showed enhanced IDO1 expression, while no change in either IDO1 or -2 was found for plasmacytoid DCs. Up-regulation of IDO1 gene expression was confirmed by the presence of enhanced kynurenine/tryptophan ratios in the plasma from chronic HCV patients. Increased IDO1 and -2 expression was also observed in monocytes from healthy donors infected with an adapted mutant of the HCV JFH-1 strain ex vivo, confirming a direct effect of HCV infection. These changes in IDO expression could be prevented by treatment with the IDO inhibitor 1-methyl tryptophan (1-mT). Furthermore, maturation of monocyte-derived DCs from chronically infected HCV patients, as well as well as monocyte-derived DCs infected ex vivo with HCV, was impaired, but this was reversed by 1-mT treatment. This suggests that IDO inhibitors may be used to treat chronic HCV patients in vivo, in conjunction with current therapies, or to activate DCs from patients ex vivo, such that they can be administered back as a DC-based therapeutic vaccine.

An insight on antibody-mediated immune-metabolism in inflammation and homeostasis: the role of Fc gamma receptors in regulation of indolemaine 2,3 dioxygenase activity in dendritic cells (IRM9P.460)

Abstract Indoleamine2, 3-dioxygenase (IDO) is a key immune-modulatory enzyme in Tryptophan catabolism that is highly expressed by Dendritic cells (DCs). Notably, DCs are the main mediators of Fc-dependent anti-inflammatory effects of Intravenous Immunoglobulins (IVIgs) as well as Immune Complexes (ICs)-induced inflammation. Hence, we aimed to study the role of FcγRs in regulation of IDO activity in human DCs from healthy individuals, which remains elusive compared to the well-established role of TLRs. Plasma-pooled human IgG showed no effect on IDO activity in un-stimulated monocyte-derived DCs (Mo-DCs). However, on TLR4 stimulation, IgG down-regulates LPS-induced IDO activity. This counter-regulation was enhanced further, by blocking FcγRIIA, the highest expressed FcγR on Mo-DCs. Interestingly, this effect was elicited by both soluble and plate-bound IgG, resembling IVIgs and ICs, respectively, but with higher potency with soluble IgG. Strikingly, mouse IgG demonstrated a similar effect to human IgG. Herein, we propose a threshold model for IDO regulation through a crosstalk between TLR and FcγRs in homeostatic and inflammatory conditions. IgG was only able to down-regulates TLR-4-induced IDO activity in human DCs, with no effect in the steady state. Furthermore, data suggest that recognition of conserved proteins or glycan moieties of IgG by gamma associated receptors modulate IDO activity. Such insight has potential therapeutic implications for antibody-based immunotherapy.

Abstract P2-15-04: A phase 1/2 study of Ad.p53 DC vaccine with indoximod immunotherapy in metastatic breast cancer

Abstract Background: Indoleamine 2,3 dioxygenase (IDO) is a tryptophan-catabolizing enzyme that causes immunosuppression in the tumor microenvironment. Indoximod is an IDO pathway inhibitor. Preclinical data suggests indoximod enhancs the activity of dendritic cell (DC) vaccines. Ad.p53 is an adenovirus used to generate autologous dendritic cell (DC) vaccines against p53 epitopes. We initiated a phase 1/2a trial of indoximod + Ad.p53DC to explore the safety and efficacy of the combination along with response to subsequent chemo. The phase 1 safety data were previously presented and the treatment was well tolerated with no DLTs. (Soliman, ASCO 2013) This abstract includes new phase 2a safety/efficacy data and updated outcomes on all phase 1/2 metastatic breast cancer patients who received Adp53DC+indoximod and any subsequent response to salvage chemo. Methods: The phase 2a study combined indoximod 1600mg PO BID with up to 6 Ad.p53 DC vaccinations q2wks. The trial used a single arm, Simon two stage design (n=12 in 1st stage, 25 in 2nd stage) with objective response as the primary endpoint. One response out of 12 was required for progression into second stage. The study had 90% power to detect 20% response rate with a p=.09. Patients with measurable, metastatic breast cancer, &amp;lt;3 lines of chemo in metastatic setting, p53 IHC &amp;gt;5%, ECOG 0-2, no autoimmune disease were eligible. Study treatment continued until disease progression or unacceptably toxicity. Results: Twelve phase 2 patients were accrued, 9 (7 TNBC, 2 ER+/HER2-) received ≥ 1 dose of Ad.p53DC+indoximod (3 did not due to rapid disease progression during vaccine preparation). Six patients had ≥ 1 prior line of chemo. Seven (58%) subjects experienced any grade AE, there were no treatment related AEs ≥G3. All treatment attributable AEs were G1-2, &amp;lt;10% frequency, and included anemia, nausea, lymphopenia, photophobia, and headache. All discontinuations were due to disease progression. Best response to immunotherapy in phase 2=1 SD, 8 PD. Phase 2 median TTP = 6.85 wks (3.8-18.1), OS = 18.1 wks (3.8-52) with 3 patients alive as of June 2014. Five patients who received ≥ 2 cycles of chemotherapy after immunotherapy demonstrated 1 PR, 3 PD, 1 pending scan. For the entire phase 1/2 breast cohort (21 in phase 1, 9 in phase 2) the median TTP = 9.85 weeks (3.8-22.1), OS=38.7 (3.8-122.1) weeks, with 1 patient from the phase 1 cohort alive as of June 2014. Ten out of 21 evaluable patients (1 CR, 7 PR, 2 SD, 11 PD) had clinical benefit from chemotherapy after immunotherapy (9 patients had rapid decline and did not get one full cycle of therapy). All but one of the responders had seen prior chemotherapy in the metastatic setting (including a CR after 4 prior lines of chemo). Median OS in the chemo responders was 69.4 wks (30.1-122.1). Conclusions: Indoximod+Ad.p53DC was well tolerated. Across phase 1/2 the best response to immunotherapy alone was SD in 4 pts while 10 of 21 (47%) (including 1 CR) responded to subsequent chemotherapy in this largely pretreated cohort. There may be a chemosensitization effect of indoximod+Ad.p53DC. Future trials should combine this treatment with chemotherapy in appropriately selected patients. Citation Format: Hatem H Soliman, Susan E Minton, Roohi Ismail-Khan, Hyo S Han, Nicholas N Vahanian, Charles J Link, Gene Kennedy, Howard Streicher, Daniel Sullivan, Scott J Antonia. A phase 1/2 study of Ad.p53 DC vaccine with indoximod immunotherapy in metastatic breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-15-04.

Accumulation of an endogenous tryptophan-derived metabolite in colorectal and breast cancers.

Tumor immune escape mechanisms are being regarded as suitable targets for tumor therapy. Among these, tryptophan catabolism plays a central role in creating an immunosuppressive environment, leading to tolerance to potentially immunogenic tumor antigens. Tryptophan catabolism is initiated by either indoleamine 2,3-dioxygenase (IDO-1/-2) or tryptophan 2,3-dioxygenase 2 (TDO2), resulting in biostatic tryptophan starvation and l-kynurenine production, which participates in shaping the dynamic relationship of the host’s immune system with tumor cells. Current immunotherapy strategies include blockade of IDO-1/-2 or TDO2, to restore efficient antitumor responses. Patients who might benefit from this approach are currently identified based on expression analyses of IDO-1/-2 or TDO2 in tumor tissue and/or enzymatic activity assessed by kynurenine/tryptophan ratios in the serum. We developed a monoclonal antibody targeting l-kynurenine as an in situ biomarker of IDO-1/-2 or TDO2 activity. Using Tissue Micro Array technology and immunostaining, colorectal and breast cancer patients were phenotyped based on l-kynurenine production. In colorectal cancer l-kynurenine was not unequivocally associated with IDO-1 expression, suggesting that the mere expression of tryptophan catabolic enzymes is not sufficiently informative for optimal immunotherapy.