The tryptophan-degrading enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a plastic immune checkpoint molecule that potently orchestrates immune responses within the tumor microenvironment (TME). As a heme-containing protein, IDO1 catalyzes the conversion of the essential amino acid tryptophan into immunoactive metabolites, called kynurenines. By depleting tryptophan and enriching the TME with kynurenines, IDO1 catalytic activity shapes an immunosuppressive TME. Accordingly, the inducible or constitutive IDO1 expression in cancer correlates with a negative prognosis for patients, representing one of the critical tumor-escape mechanisms. However, clinically trialed IDO1 catalytic inhibitors disappointed the expected anti-tumor efficacy. Interestingly, the non-enzymatic apo-form of IDO1 is still active as a transducing protein, capable of promoting an immunoregulatory phenotype in dendritic cells (DCs) as well as a pro-tumorigenic behavior in murine melanoma. Moreover, the IDO1 catalytic inhibitor epacadostat can induce a tolerogenic phenotype in plasmacytoid DCs, overcoming the catalytic inhibition of IDO1. Based on this recent evidence, IDO1 plasticity was investigated in the human ovarian cancer cell line, SKOV-3, that constitutively expresses IDO1 in a dynamic balance between the holo- and apo-protein, and thus potentially endowed with a dual function (i.e., enzymatic and non-enzymatic). Besides inhibiting the catalytic activity, epacadostat persistently stabilizes the apo-form of IDO1 protein, favoring its tyrosine-phosphorylation and promoting its association with the phosphatase SHP-2. In SKOV-3 cells, both these early molecular events activate a signaling pathway transduced by IDO1 apo-protein, which is independent of its catalytic activity and contributes to the tumorigenic phenotype of SKOV-3 cells. Overall, our findings unveiled a new mechanism of action of epacadostat on IDO1 target, repositioning the catalytic inhibitor as a stabilizer of the apo-form of IDO1, still capable of transducing a pro-tumorigenic pathway in SKOV-3 tumor. This mechanism could contribute to clarify the lack of effectiveness of epacadostat in clinical trials and shed light on innovative immunotherapeutic strategies to tackle IDO1 target.

Small synthetic oligodeoxynucleotides (ODNs) can mimic microbial nucleic acids by interacting with receptor systems and promoting immunostimulatory activities. Nevertheless, some ODNs can act differently on the plasmacytoid dendritic cell (pDC) subset, shaping their immunoregulatory properties and rendering them suitable immunotherapeutic tools in several clinical settings for treating overwhelming immune responses. We designed HIV–1–derived, DNA- and RNA-based oligonucleotides (gag, pol, and U5 regions) and assessed their activity in conferring a tolerogenic phenotype to pDCs in skin test experiments. RNA-but not DNA-oligonucleotides are capable of inducing tolerogenic features in pDCs. Interestingly, sensing the HIV–1–derived single-stranded RNA-gag oligonucleotide (RNA-gag) requires both TLR3 and TLR7 and the engagement of the TRIF adaptor molecule. Moreover, the induction of a suppressive phenotype in pDCs by RNA-gag is contingent upon the induction and activation of the immunosuppressive enzyme Arginase 1. Thus, our data suggest that sensing of the synthetic RNA-gag oligonucleotide in pDCs can induce a suppressive phenotype in pDCs, a property rendering RNA-gag a potential tool for therapeutic strategies in allergies and autoimmune diseases.

Src is a protein tyrosine kinase commonly activated downstream of transmembrane receptors and plays key roles in cell growth, migration, and survival signaling pathways. In conventional dendritic cells (cDCs), Src is involved in the activation of the non-enzymatic functions of indoleamine 2,3-dioxygenase 1 (IDO1), an immunoregulatory molecule endowed with both catalytic activity and signal transducing properties. Prompted by the discovery that the metabolite spermidine confers a tolerogenic phenotype on cDCs that is dependent on both the expression of IDO1 and the activity of Src kinase, we here investigated the spermidine mode of action. We found that spermidine directly binds Src in a previously unknown allosteric site located on the backside of the SH2 domain and thus acts as a positive allosteric modulator of the enzyme. Besides confirming that Src phosphorylates IDO1, here we showed that spermidine promotes the protein-protein interaction of Src with IDO1. Overall, this study may pave the way toward the design of allosteric modulators able to switch on/off the Src-mediated pathways, including those involving the immunoregulatory protein IDO1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme chronically activated in many cancer patients and its expression and activity correlate with a poor prognosis. In fact, it acts as an immune regulator and contributes to tumor-induced immunosuppression by determining tryptophan deprivation and producing immunosuppressive metabolites named kynurenines. These findings made IDO1 an attractive target for cancer immunotherapy and small-molecule inhibitors, such as epacadostat, have been developed to block its enzymatic activity. Although epacadostat was effective in preclinical models and in early phase trials, it gave negative results in a metastatic melanoma randomized phase III study to test the benefit of adding epacadostat to the reference pembrolizumab therapy. However, the reason for the epacadostat failure in this clinical trial has never been understood. Our data suggest that a possible explanation of epacadostat ineffectiveness may rely on the ability of this drug to enhance the other IDO1 immunoregulatory mechanism, involving intracellular signaling function. These findings open up a new perspective for IDO1 inhibitors developed as new anticancer drugs, which should be carefully evaluated for their ability to block not only the catalytic but also the signaling activity of IDO1.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a paralog of Indoleamine 2,3-dioxygenase 1 (IDO1), a tryptophan-degrading enzyme producing immunomodulatory molecules. However, the two proteins are unlikely to carry out the same functions. IDO2 shows little or no tryptophan catabolic activity and exerts contrasting immunomodulatory roles in a context-dependent manner in cancer and autoimmune diseases. The recently described potential non-enzymatic activity of IDO2 has suggested its possible involvement in alternative pathways, resulting in either pro- or anti-inflammatory effects in different models. In a previous study on non-small cell lung cancer (NSCLC) tissues, we found that IDO2 expression revealed at the plasma membrane level of tumor cells was significantly associated with poor prognosis. In this study, the A549 human cell line, basally expressing IDO2, was used as an in vitro model of human lung adenocarcinoma to gain more insights into a possible alternative function of IDO2 different from the catalytic one. In these cells, immunocytochemistry and isopycnic sucrose gradient analyses confirmed the IDO2 protein localization in the cell membrane compartment, and the immunoprecipitation of tyrosine-phosphorylated proteins revealed that kinase activities can target IDO2. The different localization from the cytosolic one and the phosphorylation state are the first indications for the signaling function of IDO2, suggesting that the IDO2 non-enzymatic role in cancer cells is worthy of deeper understanding.

Over the last two decades, indoleamine 2,3-dioxygenase 1 (IDO1) has attracted wide interest as a key player in immune regulation, fostering the design and development of small molecule inhibitors to restore immune response in tumor immunity. In this framework, biochemical, structural, and pharmacological studies have unveiled peculiar structural plasticity of IDO1, with different conformations and functional states that are coupled to fine regulation of its catalytic activity and non-enzymic functions. The large plasticity of IDO1 may affect its ligand recognition process, generating bias in structure-based drug design campaigns. In this work, we report a screening campaign of a fragment library of compounds, grounding on the use of three distinct conformations of IDO1 that recapitulate its structural plasticity to some extent. Results are instrumental to discuss tips and pitfalls that, due to the large plasticity of the enzyme, may influence the identification of novel and differentiated chemical scaffolds of IDO1 ligands in structure-based screening campaigns.

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the initial rate-limiting step in the degradation of the essential amino acid tryptophan along the kynurenine pathway. When discovered more than 50 years ago, IDO1 was thought to be an effector molecule capable of mediating a survival strategy based on the deprivation of bacteria and tumor cells of the essential amino acid tryptophan. Since 1998, when tryptophan catabolism was discovered to be crucially involved in the maintenance of maternal T-cell tolerance, IDO1 has become the focus of several laboratories around the world. Indeed, IDO1 is now considered as an authentic immune regulator not only in pregnancy, but also in autoimmune diseases, chronic inflammation, and tumor immunity. However, in the last years, a bulk of new information-including structural, biological, and functional evidence-on IDO1 has come to light. For instance, we now know that IDO1 has a peculiar conformational plasticity and, in addition to a complex and highly regulated catalytic activity, is capable of performing a nonenzymic function that reprograms the expression profile of immune cells toward a highly immunoregulatory phenotype. With this state-of-the-art review, we aimed at gathering the most recent information obtained for this eclectic protein as well as at highlighting the major unresolved questions.

The study of kinetics and thermodynamics of protein-protein interactions can contribute to assessment of the mechanism of molecular recognition process. These analyses can provide information about conformational changes and noncovalent forces that influence the initial recognition between proteins and stabilization of the complex. Studying these aspects may lead to a better comprehension of functions of proteins in biological environment and can become useful for the rational modification of some interactions by engineering of one of the implicated partners. Real-time biosensor assays based on surface plasmon resonance have been widely applied for the label-free evaluation of protein-protein interactions, allowing their characterization in term of binding affinity and kinetics. In the present chapter, we provide a protocol for the assessment of interactions involving complement proteins or antibodies, the protagonists of the immune system. We reported guidelines and indications concerning the analysis of the experimental data for the estimation of the kinetic parameters and for the evaluation of activation and equilibrium binding thermodynamics.

Immunoglobulin repertoires contain a fraction of antibodies that recognize low molecular weight compounds, including some enzymes’ cofactors, such as heme. Here, by using a set of 113 samples with variable region sequences matching clinical-stage antibodies, we demonstrated that a considerable number of these antibodies interact with heme. Antibodies that interact with heme possess specific sequence traits of their antigen-binding regions. Moreover they manifest particular physicochemical and functional qualities i.e. increased hydrophobicity, higher propensity of self-binding, higher intrinsic polyreactivity and reduced expression yields. Thus, interaction with heme is a strong predictor of different molecular and functional qualities of antibodies. Notably, these qualities are of high importance for therapeutic antibodies, as their presence was associated with failure of drug candidates to reach clinic. Our study reveled an important facet of information about relationship sequence-function in antibodies. It also offers a convenient tool for detection of liabilities of therapeutic antibodies.

Polyamines (i.e., putrescine, spermidine, and spermine) are bioactive polycations capable of binding nucleic acids and proteins and modulating signaling pathways. Polyamine functions have been studied most extensively in tumors, where they can promote cell transformation and proliferation. Recently, spermidine was found to exert protective effects in an experimental model of multiple sclerosis (MS) and to confer immunoregulatory properties on dendritic cells (DCs), via the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme. IDO1 converts l-tryptophan into metabolites, collectively known as kynurenines, endowed with several immunoregulatory effects via activation of the arylhydrocarbon receptor (AhR). Because AhR activation increases polyamine production, the emerging scenario has identified polyamines and kynurenines as actors of an immunoregulatory circuitry with potential implications for immunotherapy in autoimmune diseases and cancer.

Neonatal Fc receptor (FcRn) has a key role in the homeostasis of IgG. Despite its physiological and clinical importance, the interaction of IgG and FcRn remains not completely comprehended. Thus, IgG molecules with identical constant portions but with minor differences in their V regions have been demonstrated to interact with FcRn with a considerable heterogeneity in the binding affinity. To understand this discrepancy, we dissected the physicochemical mechanism of the interaction of 10 human IgG1 to human FcRn. The interactions of two Abs in the presence of their cognate Ags were also examined. Data from activation and equilibrium thermodynamics analyses as well as pH dependence of the kinetics revealed that the V region of IgG could modulate a degree of conformational changes and binding energy of noncovalent contacts at the FcRn binding interface. These results suggest that the V domains modulate FcRn binding site in Fc by allosteric effects. These findings contribute for a deeper understanding of the mechanism of IgG-FcRn interaction. They might also be of relevance for rational engineering of Abs for optimizing their pharmacokinetic properties.

Therapeutic intravenous immunoglobulin preparations (IVIg) are used for treatment of wide range of autoimmune and inflammatory diseases. Versatile mechanisms have been reported to contribute to the immunomodulatory effects of IVIg. Here we demonstrate that IVIg has a strong potential to inhibit pro-inflammatory effect of extracellular heme. Indeed, the presence of immunoglobulins reduced the potential of heme to activate the complement system on the surface of human endothelial cells. Since extracellular heme is considered as one of the principal pathogenic factors in hemolytic disorders, its therapeutic scavenging by IVIg may have significant clinical repercussions.

OBJECTIVES This Report intends to estimate the total number of people still alive in 2010 after cancer diagnosis in Italy, regardless of the time since diagnosis, and to project these estimates to 2015. This study is also aimed to estimate the number of already cured cancer patients, whose mortality rates have become undistinguishable from that of the general population of the same age and sex. MATERIALS AND METHODS The study took advantage of the information from the AIRTUM database, which included 29 Cancer Registries (covering 21 million people, 35% of the Italian population). A total of 1,624,533 cancer cases diagnosed between 1976 and 2009 contributed to the study. For each registry, the observed prevalence was calculated. Prevalence for lengths of time exceeding the maximum duration of the registration and of the complete prevalence were derived by applying an estimated correction factor, the completeness index. This index was estimated by means of statistical regression models using cancer incidence and survival data available in registries with 18 years of observation or more. For 50 types or combinations of neoplasms, complete prevalence was estimated at 1.1.2010 as an absolute number and as a proportion per 100,000 inhabitants by sex, age group, area of residence, and years since diagnosis. Projections of complete prevalence for 1.1.2015 were computed under the assumption of a linear trend of the complete prevalence observed until 2010. Validated mixture cure models were used to estimate: the cure fraction, that is the proportion of patients who, starting from the time of diagnosis, are expected to reach the same mortality rate of the general population; the conditional relative survival (CRS), that is the cumulative probability of surviving some additional years, given that patients already survived a certain number of years; the time to cure, that is the number of years necessary so that conditional survival in the following five years (5-year CRS) exceeds the conventional threshold of 95% (i.e., mortality rates in cancer patients become undistinguishable compared to those of the general population); the proportion of patients already cured, i.e., people alive since a number of years exceeding time to cure. RESULTS As of 1.1.2010, it was estimated that 2,587,347 people were alive after a cancer diagnosis, corresponding to 4.4% of the Italian population. A relevant geographical heterogeneity emerged, with a prevalence above 5% in northern registries and below 4% in southern areas. Men were 45% of the total (1,154,289) and women 55% (1,433,058). In the population aged 75 years or more, the proportions of prevalent cases were 20% in males and 13% in females, 11% between 60 and 74 years of age in both sexes. Nearly 600,000 Italian women were alive after a breast cancer diagnosis (41% of all women with this neoplasm), followed by women with cancers of the colon rectum (12%), corpus uteri (7%), and thyroid (6%). In men, 26% of prevalent cases (295,624) were patients with prostate cancer, 16% with either bladder or colon rectum cancer. The projections for 1.1.2015 are of three million (3,036,741) people alive after a cancer diagnosis, 4.9% of the Italian population; with a 20% increase for males and 15% for females, compared to 2010. The cure fractions were heterogeneous according to cancer type and age. Estimates obtained as the sum of cure fractions for all cancer types showed that more than 60% of patients diagnosed below the age of 45 years will reach the same mortality rate of the general population. This proportion decreased with increasing age and it was <30% for cancer diagnosed after the age of 74 years. It was observed that 60% of all prevalent cases (1,543,531 people or 2.6% of overall Italian population) had been diagnosed >5 years earlier (long-term survivors). Time to cure (5-year CRS>95%) was reached in <10 years by patients with cancers of the stomach, colon rectum, pancreas, corpus and cervix uteri, brain, and Hodgkin lymphoma. Mortality rates similar to the ones reported by the general population were reached after approximately 20 years for breast and prostate cancer patients. Five-year CRS remained <95% for >25 years after cancer diagnosis in patients with liver and larynx cancers, non-Hodgkin lymphoma, myeloma, and leukaemia. Time to cure was reached by 27% (20% in men and 33% in women) of all people living after a cancer diagnosis, defined as already cured. CONCLUSIONS The study showed a steady increase over time (nearly +3% per year) of prevalent cases in Italy. A quarter of Italian cancer patients alive in 2010 can be considered as already cured. The AIRTUM Report 2014 describes characteristics of cancer patients and former-patients for 50 cancer types or combinations by sex and age. This detailed information promotes the conduction of studies aimed at expanding the current knowledge on the quality of life of these patients during and after the active phase of treatments (prevalence according to health status), on the long-term effects of treatments (in particular for paediatric patients), on the cost profile of cancer patients, and on rare tumours. All these observations have a high potential impact on health planning, clinical practice, and, most of all, patients' perspective.