Indoximod opposes the immunosuppressive effects mediated by IDO and TDO via modulation of AhR function and activation of mTORC1.

Indoximod has shaped our understanding of the biology of IDO1 in the control of immune responses, though its mechanism of action has been poorly understood. Previous studies demonstrated that indoximod creates a tryptophan (Trp) sufficiency signal that reactivates mTOR in the context of low Trp concentrations, thus opposing the effects caused by IDO1. Here we extend the understanding of indoximod's mechanism of action by showing that it has pleiotropic effects on immune regulation. Indoximod can have a direct effect on T cells, increasing their proliferation as a result of mTOR reactivation. Further, indoximod modulates the differentiation of CD4+ T cells via the aryl hydrocarbon receptor (AhR), which controls transcription of several genes in response to different ligands including kynurenine (Kyn). Indoximod increases the transcription of RORC while inhibiting transcription of FOXP3, thus favoring differentiation to IL-17-producing helper T cells and inhibiting the differentiation of regulatory T cells. These indoximod-driven effects on CD8+ and CD4+ T cells were independent from the activity of IDO/TDO and from the presence of exogenous Kyn, though they do oppose the effects of Kyn produced by these Trp catabolizing enzymes. Indoximod can also downregulate expression of IDO protein in vivo in murine lymph node dendritic cells and in vitro in human monocyte-derived dendritic cells via a mechanism that involves signaling through the AhR. Together, these data improve the understanding of how indoximod influences the effects of IDO, beyond and distinct from direct enzymatic inhibition of the enzyme.

Discovery of indoximod prodrugs and characterization of clinical candidate NLG802.

A series of different prodrugs of indoximod, including estesrs and peptide amides were synthesized with the aim of improving its oral bioavailability in humans. The pharmacokinetics of prodrugs that were stable in buffers, plasma and simulated gastric and intestinal fluids was first assessed in rats after oral dosing in solution or in capsule formulation. Two prodrugs that produced the highest exposure to indoximod in rats were further tested in Cynomolgus monkeys, a species in which indoximod has oral bioavailability of 6-10% and an equivalent dose-dependent exposure profile as humans. NLG802 was selected as the clinical development candidate after increasing oral bioavailability (>5-fold), Cmax (6.1-3.6 fold) and AUC (2.9-5.2 fold) in monkeys, compared to equivalent molar oral doses of indoximod. NLG802 is extensively absorbed and rapidly metabolized to indoximod in all species tested and shows a safe toxicological profile at the anticipated therapeutic doses. NLG802 markedly enhanced the anti-tumor responses of tumor-specific pmel-1 T cells in a melanoma tumor model. In conclusion, NLG802 is a prodrug of indoximod expected to increase clinical drug exposure to indoximod above the current achievable levels, thus increasing the possibility of therapeutic effects in a larger fraction of the target patient population.

Implementation of the CYP Index for the Design of Selective Tryptophan-2,3-dioxygenase Inhibitors.

A class of imidazoisoindole (III) heme-binding indoleamine-2,3-dioxygenase (IDO1) inhibitors were optimized via structure-based drug design into a series of tryptophan-2,3-dioxygenase (TDO)-selective inhibitors. Kynurenine pathway modulation was demonstrated in vivo, which enabled evaluation of TDO as a potential cancer immunotherapy target. As means of mitigating the risk of drug-drug interactions arising from cytochrome P450 inhibition, a novel property-based drug design parameter, herein referred to as the CYP Index, was implemented for the design of inhibitors with appreciable selectivity for TDO over CYP3A4. We anticipate the CYP Index will be a valuable design parameter for optimizing CYP inhibition of any small molecule inhibitor containing a Lewis basic motif capable of binding heme.

Discovery of Clinical Candidate (1R,4r)-4-((R)-2-((S)-6-Fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)cyclohexan-1-ol (Navoximod), a Potent and Selective Inhibitor of Indoleamine 2,3-Dioxygenase 1.

A novel class of 5-substituted 5H-imidazo[5,1-a]isoindoles are described as potent inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1). A structure-based drug design approach was used to elaborate the 5H-imidazo[5,1-a]isoindole core and to improve potency and pharmacological properties. Suitably placed hydrophobic and polar functional groups in the lead molecule allowed improvement of IDO1 inhibitory activity while minimizing off-target liabilities. Structure-activity relationship studies focused on optimizing IDO1 inhibition potency and a pharmacokinetic profile amenable to oral dosing while controlling CYP450 and hERG inhibitory properties.

Phase Ia study of the indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor navoximod (GDC-0919) in patients with recurrent advanced solid tumors.

BACKGROUND: Indoleamine-2,3-dioxygenase 1 (IDO1) catalyzes the oxidation of tryptophan into kynurenine and is partially responsible for acquired immune tolerance associated with cancer. The IDO1 small molecule inhibitor navoximod (GDC-0919, NLG-919) is active as a combination therapy in multiple tumor models. METHODS: This open-label Phase Ia study assessed safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary anti-tumor activity of navoximod in patients with recurrent/advanced solid tumors, administered as 50-800 mg BID on a 21/28 day and at 600 mg on a 28/28 day schedule. Plasma kynurenine and tryptophan were longitudinally evaluated and tumor assessments were performed. RESULTS: Patients (n = 22) received a median of 3 cycles of navoximod. No maximum tolerated dose was reached. One dose-limiting toxicity of Grade 4 lower gastrointestinal hemorrhage was reported. Adverse events (AEs) regardless of causality in ≥20% of patients included fatigue (59%), cough, decreased appetite, and pruritus (41% each), nausea (36%), and vomiting (27%). Grade ≥ 3 AEs occurred in 14/22 patients (64%), and were related to navoximod in two patients (9%). Navoximod was rapidly absorbed (Tmax ~ 1 h) and exhibited dose-proportional increases in exposure, with a half-life (t1/2 ~ 11 h) supportive of BID dosing. Navoximod transiently decreased plasma kynurenine from baseline levels with kinetics consistent with its half-life. Of efficacy-evaluable patients, 8 (36%) had stable disease and 10 (46%) had progressive disease. CONCLUSIONS: Navoximod was well-tolerated at doses up to 800 mg BID decreasing plasma kynurenine levels consistent with its half-life. Stable disease responses were observed. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02048709 .

A phase I study of indoximod in patients with advanced malignancies.

PURPOSE: Indoximod is an oral inhibitor of the indoleamine 2,3-dioxygenase pathway, which causes tumor-mediated immunosuppression. Primary endpoints were maximum tolerated dose (MTD) and toxicity for indoximod in patients with advanced solid tumors. Secondary endpoints included response rates, pharmacokinetics, and immune correlates. EXPERIMENTAL DESIGN: Our 3+3 phase I trial comprised 10 dose levels (200, 300, 400, 600, and 800 mg once/day; 600, 800, 1200, 1600, and 2000 mg twice/day). Inclusion criteria were measurable metastatic solid malignancy, age ≥18 years, and adequate organ/marrow function. Exclusion criteria were chemotherapy ≤ 3 weeks prior, untreated brain metastases, autoimmune disease, or malabsorption. RESULTS: In 48 patients, MTD was not reached at 2000 mg twice/day. At 200 mg once/day, 3 patients previously treated with checkpoint inhibitors developed hypophysitis. Five patients showed stable disease >6 months. Indoximod plasma AUC and Cmax plateaued above 1200mg. Cmax (~12 µM at 2000 mg twice/day) occurred at 2.9 hours, and half-life was 10.5 hours. C reactive protein (CRP) levels increased across multiple dose levels. CONCLUSIONS: Indoximod was safe at doses up to 2000 mg orally twice/day. Best response was stable disease >6 months in 5 patients. Induction of hypophysitis, increased tumor antigen autoantibodies and CRP levels were observed.

The PTEN pathway in Tregs is a critical driver of the suppressive tumor microenvironment.

The tumor microenvironment is profoundly immunosuppressive. We show that multiple tumor types create intratumoral immune suppression driven by a specialized form of regulatory T cell (Treg) activation dependent on the PTEN (phosphatase and tensin homolog) lipid phosphatase. PTEN acted to stabilize Tregs in tumors, preventing them from reprogramming into inflammatory effector cells. In mice with a Treg-specific deletion of PTEN, tumors grew slowly, were inflamed, and could not create an immunosuppressive tumor microenvironment. In normal mice, exposure to apoptotic tumor cells rapidly elicited PTEN-expressing Tregs, and PTEN-deficient mice were unable to maintain tolerance to apoptotic cells. In wild-type mice with large established tumors, pharmacologic inhibition of PTEN after chemotherapy or immunotherapy profoundly reconfigured the tumor microenvironment, changing it from a suppressive to an inflammatory milieu, and tumors underwent rapid regression. Thus, the immunosuppressive milieu in tumors must be actively maintained, and tumors become susceptible to immune attack if the PTEN pathway in Tregs is disrupted.

IDO inhibits a tryptophan sufficiency signal that stimulates mTOR: A novel IDO effector pathway targeted by D-1-methyl-tryptophan.

Tryptophan catabolism by indoleamine 2,3-dioxygenase (IDO) alters inflammation and favors T-cell tolerance in cancer, but the underlying molecular mechanisms remain poorly understood. The integrated stress response kinase GCN2, a sensor of uncharged tRNA that is activated by amino acid deprivation, is recognized as an important effector of the IDO pathway. However, in a mouse model of inflammatory carcinogenesis, ablation of Gcn2 did not promote resistance against tumor development like the absence of IDO does, implying the existence of additional cancer-relevant pathways that operate downstream of IDO. Addressing this gap in knowledge, we report that the IDO-mediated catabolism of tryptophan also inhibits the immunoregulatory kinases mTOR and PKC-Θ, along with the induction of autophagy. These effects were relieved specifically by tryptophan but also by the experimental agent 1-methyl-D-tryptophan (D-1MT, also known as NLG8189), the latter of which reversed the inhibitory signals generated by IDO with higher potency. Taken together, our results implicate mTOR and PKC-Θ in IDO-mediated immunosuppressive signaling, and they provide timely insights into the unique mechanism of action of D-1MT as compared with traditional biochemical inhibitors of IDO. These findings are important translationally, because they suggest broader clinical uses for D-1MT against cancers that overexpress any tryptophan catabolic enzyme (IDO, IDO2 or TDO). Moreover, they define mTOR and PKC-Θ as candidate pharmacodynamic markers for D-1MT responses in patients recruited to ongoing phase IB/II cancer trials, addressing a current clinical need.

Amino acid catabolism: a pivotal regulator of innate and adaptive immunity.

Enhanced amino acid catabolism is a common response to inflammation, but the immunologic significance of altered amino acid consumption remains unclear. The finding that tryptophan catabolism helped maintain fetal tolerance during pregnancy provided novel insights into the significance of amino acid metabolism in controlling immunity. Recent advances in identifying molecular pathways that enhance amino acid catabolism and downstream mechanisms that affect immune cells in response to inflammatory cues support the notion that amino acid catabolism regulates innate and adaptive immune cells in pathologic settings. Cells expressing enzymes that degrade amino acids modulate antigen-presenting cell and lymphocyte functions and reveal critical roles for amino acid- and catabolite-sensing pathways in controlling gene expression, functions, and survival of immune cells. Basal amino acid catabolism may contribute to immune homeostasis that prevents autoimmunity, whereas elevated amino acid catalytic activity may reinforce immune suppression to promote tumorigenesis and persistence of some pathogens that cause chronic infections. For these reasons, there is considerable interest in generating novel drugs that inhibit or induce amino acid consumption and target downstream molecular pathways that control immunity. In this review, we summarize recent developments and highlight novel concepts and key outstanding questions in this active research field.

Altered tryptophan metabolism as a paradigm for good and bad aspects of immune privilege in chronic inflammatory diseases.

The term "immune privilege" was coined to describe weak immunogenicity (hypo-immunity) that manifests in some transplant settings. We extended this concept to encompass hypo-immunity that manifests at local sites of inflammation relevant to clinical diseases. Here, we focus on emerging evidence that enhanced tryptophan catabolism is a key metabolic process that promotes and sustains induced immune privilege, and discuss the implications for exploiting this knowledge to improve treatments for hypo-immune and hyper-immune syndromes using strategies to manipulate tryptophan metabolism.

Zinc protoporphyrin IX stimulates tumor immunity by disrupting the immunosuppressive enzyme indoleamine 2,3-dioxygenase.

The tryptophan catabolic enzyme indoleamine 2,3-dioxygenase (IDO) has emerged as an important driver of immune escape in a growing number of cancers and cancer-associated chronic infections. In this study, we define novel immunotherapeutic applications for the heme precursor compound zinc protoporphyrin IX (ZnPP) based on our discovery that it is a potent small-molecule inhibitor of IDO. Inhibitory activity was determined using in vitro and in-cell enzyme assays as well as a novel in vivo pharmacodynamic system. An irreversible mechanism of inhibition was documented, consistent with competition for heme binding in newly synthesized cellular protein. siRNA methodology and an IDO-deficient mouse strain were used to verify the specificity of ZnPP as an IDO inhibitor. In a preclinical model of melanoma, ZnPP displayed antitumor properties that relied on T-cell function and IDO integrity. ZnPP also phenocopied the known antitumor properties of IDO inhibitors in preclinical models of skin and breast carcinoma. Our results suggest clinical evaluation of ZnPP as an adjuvant immunochemotherapy in chronic infections and cancers in which there is emerging recognition of a pathophysiologic role for IDO dysregulation.

Allogeneic melanoma vaccine expressing alphaGal epitopes induces antitumor immunity to autologous antigens in mice without signs of toxicity.

Owing to the absence of alphaGal epitopes in human cells and constant stimulation of the immune system by the symbiotic bacterial flora, humans develop high titers of natural antibodies against these epitopes. It has been demonstrated that syngeneic whole cell vaccines modified to express alphaGal epitopes could be used to generate a potent anticancer vaccine. In this study, we tested whether allogeneic whole cell cancer vaccines modified to express alphaGal epitopes would be effective for the treatment of murine melanoma. The alpha(1,3)galactosyltransferase (alphaGT) knockout mice (H-2) with preexisting subcutaneous and pulmonary tumors [alphaGal B16, H-2] received therapeutic vaccinations with S91M3alphaGal (H-2) whole cell allogeneic vaccines. These mice had better survival and reduced pulmonary metastasis burden compared with control mice treated with S91M3 vaccine cells. Vaccination with S91M3alphaGal-induced cytotoxic CD8 T cells recognizing the syngeneic alphaGal B16 tumors measured by adoptive transfer to recipients bearing pulmonary metastases. The presence of allo-antigens did not dominate the induction of immunity to "cryptic" tumor antigens and had helped in the generation of a more efficient vaccine to treat preexisting tumors when compared with classic autologous vaccines. Vaccination with allogeneic alphaGal vaccines did not induce signs of toxicity including changes in weight, hematology, chemistry, and histopathology of major perfused organs or autoimmunity in long-term murine models for breast, lung, and melanoma. This study established the safety and efficacy data of allogeneic alphaGal whole cell vaccines and constituted the basis for the initiation of human clinical trials to treat human malignancies.

Effective treatment of preexisting melanoma with whole cell vaccines expressing alpha(1,3)-galactosyl epitopes.

The hyperacute immune response in humans is a potent mechanism of xenograft rejection mediated by complement-fixing natural antibodies recognizing alpha(1,3)-galactosyl epitopes (alphaGal) not present on human cells. We exploited this immune mechanism to create a whole cell cancer vaccine to treat melanoma tumors. B16 melanoma vaccines genetically engineered to express alphaGal epitopes (B16alphaGal) effectively treated preexisting s.c. and pulmonary alphaGal-negative melanoma (B16Null) tumors in the alpha(1,3)-galactosyltransferase knockout mouse model. T cells from mice vaccinated with B16alphaGal recognized B16Null melanoma cells measured by detection of intracellular tumor necrosis factor-alpha. We showed successful adoptive transfer of immunity to recipient mice bearing lung melanoma metastasis. Mice receiving lymphocytes from donors previously immunized with B16alphaGal had reduced pulmonary metastases. The transfer of lymphocytes from mice vaccinated with control vaccine had no effect in the pulmonary metastasis burden. This study unequivocally establishes for the first time efficacy in the treatment of preexisting melanoma tumors using whole cell vaccines expressing alphaGal epitopes. Vaccination with B16alphagal induced strong long-lasting cell-mediated antitumor immunity extended to B16Null. These data formed the basis for the testing of this therapeutic strategy in human clinical trials currently under way.

In vivo levels of S-adenosylmethionine modulate C:G to T:A mutations associated with repeat-induced point mutation in Neurospora crassa.

In Neurospora crassa, the mutagenic process termed repeat-induced point mutation (RIP) inactivates duplicated DNA sequences during the sexual cycle by the introduction of C:G to T:A transition mutations. In this work, we have used a collection of N. crassa strains exhibiting a wide range of cellular levels of S-adenosylmethionine (AdoMet), the universal donor of methyl groups, to explore whether frequencies of RIP are dependent on the cellular levels of this metabolite. Mutant strains met-7 and eth-1 carry mutations in genes of the AdoMet pathway and have low levels of AdoMet. Wild type strains with high levels of AdoMet were constructed by introducing a chimeric transgene of the AdoMet synthetase (AdoMet-S) gene fused to the constitutive promoter trpC from Aspergillus nidulans. Crosses of these strains against tester duplications of the pan-2 and am genes showed that frequencies of RIP, as well as the total number of C:G to T:A transition mutations found in randomly selected am(RIP) alleles, are inversely correlated to the cellular level of AdoMet. These results indicate that AdoMet modulates the biochemical pathway leading to RIP.

High-efficiency lentiviral vector-mediated gene transfer into murine macrophages and activated splenic B lymphocytes.

The goal of the present report was to determine if lentiviral vectors could mediate gene transfer into murine terminally differentiated macrophages and mature B lymphocytes as a new strategy of gene delivery into professional antigen-presenting cells (APC). We demonstrated that nondividing tissue resident macrophages were efficiently transduced in vitro by lentiviral vectors. Gene transfer efficiencies of up to 90% were demonstrated using a green fluorescent protein (GFP) reporter gene-containing vector and expression was stable for the length of cell culture. Transduced macrophages were functionally competent, preserving their phagocytic activity, accessory cell function, interleukin (IL)-12 secretion, and nitric oxide (NO) production similar to control untransduced macrophages. Lentiviral vector mediated transduction of CD19(+) B cell blasts was demonstrated to be in the range of 60%-70% GFP-positive cells. These transduced cells retain the ability to upregulate CD80 and CD86 similar to control B cell cultures. In addition, we show that the human immunodeficiency virus type 1 (HIV-1) accessory proteins Nef, Vpr, Vif, and Vpu are not required for the transduction of both resident macrophages and activated B lymphoblasts. We conclude that HIV-1-based lentiviral vectors can mediate efficient gene transfer into primary murine macrophages and mature B lymphocytes.

Lentiviral vectors for gene therapy of HIV-1 infection.

Lentiviral vectors based on HIV-1, HIV-2, or SIV have the ability to transduce dividing and non-dividing T cells, dendritic cells, hematopoietic stem cells and macrophages, which are the main target cells for gene therapy of HIV-1 infection. Besides their function as gene delivery vehicles, lentiviral vector backbones containing the cis-acting sequences necessary to perform a complete replication cycle in the presence of viral proteins provided in trans, have the ability to inhibit HIV-1 replication by several mechanisms that include sequestration of the regulatory proteins Tat and Rev, competition for packaging into virions and possibly by inhibition of reverse transcription in heterodimeric virions. Expression of anti-HIV-1 genes in these vectors would strengthen the potency of this inhibition. To avoid self-inhibition of the vector packaging system, lentiviral vectors have to be modified to become resistant to the anti-HIV-1 genes encoded by them. This review discusses the different genetic intervention strategies for gene therapy of HIV-1 infection focusing in the use of lentiviral vectors as the main agents to mediate inhibition of HIV-1 replication. It also discusses possible strategies to adapt HIV-1 or HIV-2 vectors to express the different classes of anti-HIV-1 genes and approaches to improve in vivo vector mobilization.

Enhanced inhibition of human immunodeficiency virus type 1 replication by novel lentiviral vectors expressing human immunodeficiency virus type 1 envelope antisense RNA.

We have developed optimized versions of a conditionally replicating human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector for gene therapy of HIV-1 infection. These vectors target HIV-1 RNAs containing sequences of the envelope gene by expressing a 1-kb fragment of the HIV-1 Tat/Rev intron in the antisense orientation. Expression of the envelope antisense gene (envAS) was evaluated under the control of different internal promoters such as the human phosphoglycerate kinase (PGK) promoter, the human EF1-alpha promoter, and the U3 region of the SL3 murine leukemia virus. The U3-SL3 promoter transactivates transcription from the vector HIV-1 LTR and drives higher expression levels of envAS-containing RNAs than other promoters in T-cell lines. The effect of other vector structural features was also evaluated. We found that the central polypurine tract and central termination sequence (cPPT) produce a small increase in vector infectivity of 2-fold to 3-fold and results in a 10-fold higher inhibition of wild-type viral replication in challenge experiments. The woodchuck hepatitis posttranscriptional regulatory element (WPRE) does not increase the cytoplasmic levels of envAS mRNA in T-cell lines. We observed that SupT1 and primary CD4(+) T cells transduced with these vectors showed high inhibition of HIV-1 replication, suppression of syncitium formation, and increased cell viability when infected with several HIV-1 laboratory strains. Our results suggest that higher vector copy number and increased levels of envAS RNA expression contribute to block replication of divergent strains of HIV-1.

Gene therapy of HIV-1 infection using lentiviral vectors expressing anti-HIV-1 genes.

The use of vectors based on primate lentiviruses for gene therapy of human immunodeficiency virus type 1 (HIV-1) infection has many potential advantages over the previous murine retroviral vectors used for delivery of genes that inhibit replication of HIV-1. First, lentiviral vectors have the ability to transduce dividing and nondividing cells that constitute the targets of HIV-1 infection such as resting T cells, dendritic cells, and macrophages. Lentiviral vectors can also transfer genes to hematopoietic stem cells with a superior gene transfer efficiency and without affecting the repopulating capacity of these cells. Second, these vectors could be potentially mobilized in vivo by the wild-type virus to secondary target cells, thus expanding the protection to previously untransduced cells. And finally, lentiviral vector backbones have the ability to block HIV-1 replication by several mechanisms that include sequestration of the regulatory proteins Tat and Rev, competition for packaging into virions, and by inhibition of reverse transcription in heterodimeric virions with possible generation of nonfunctional recombinants between the vector and viral genomes. The inhibitory ability of lentiviral vectors can be further increased by expression of anti-HIV-1 genes. In this case, the lentiviral vector packaging system has to be modified to become resistant to the anti-HIV-1 genes expressed by the vector in order to avoid self-inhibition of the vector packaging system during vector production. This review focuses on the use of lentiviral vectors as the main agents to mediate inhibition of HIV-1 replication and discusses the different genetic intervention strategies for gene therapy of HIV-1 infection.