The number of new invasive cutaneous melanoma cases projected dur ing 2010 was 68,000—a 23% increase from the 2004 prediction of 55,100 cases. In 2015, the lifetime risk of developing melanoma is estimated to increase to 1 in 50 [ Cockerell 2012 ]. Existing therapy for melanoma consists of ad equate surgical resection of the primary tumor and, in some circumstances, surgical res ection of isolated metastases. Most of the patients on the current trial will have disseminate d disease and be enroll ed after failure of standard therapies. “Little consensus exists regarding standard chemotherapy for patients with metastatic melanoma, which most likely reflects the low level of activity of older FDA approved agents” [ NCCN 2012 ]. In general, disseminated disease can be managed by systemic therapy, clinical trial or supportive care. Acco rding to NCCN Guidelines, “preferred regimens include ipilumumab, vemurafenib for patients with documented BRAF mutation, treatment in a clin ical trial and high-dose IL-2” [ NCCN 2012]. “Although approval of ipilumumab and vermurafen ib has significantly altered the initial management of patients with stage IV melanoma, each agent has unique limitations” [NCCN 2012] and might not be appropriate for all patients. Other regimens for disseminated disease “include dacarbazine, te mozolomide, imatinib for tumors with c- KIT mutations, dacarbazine- or temozolomide-based combination chemotherapy, or biochemotherapy (including cisplatin and vinbl astine with or without IL-2, interferon alfa, paclitaxel as monotherapy or in combination with carboplatin [ NCCN 2012]. These have demonstrated modest response rates under 20% in firs t-line and second-line settings.” Some of the patients on the current protocol might have in-transit metastases. According to the NCCN guidelines, “Treatment in the context of a clinical trial is the preferred option” [ NCCN 2012 ]. 2.2 CTEP and/or CIP IND Agent(s): N/A NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 9 2.3 Other Agents Indoleamine 2,3-dioxygenase IDO is an enzyme that catabolizes the essential amino acid tryptophan to kynurenine [Munn 2007; Katz 2008; Löb 2009; Soliman 2010 ] and IDO activation plays an essential role in the immunological tolera nce between mother and fetu s. In pregnant women, IDO activation decreases the concentration of tryptophan in th e blood and elevates levels of kynurenine, which, in healthy pregnancies, in creases with placental development. As a consequence, inducing IDO expression in the trophoblast suppre sses T-cell activity against the fetus [ Tatsumi 2000 ]. In murine studies, the administration of the IDO inhibitor 1-methyl-tryptophan cause d allogeneic fetal rejection [ Munn 1998]. Thus, IDO induction in the trophoblast and placenta is suffi cient to inhibit the immune system of the mother and prevent rejection of the fetus. In addition to its activation in pregnancy, IDO is also expressed by activated immune cells and inflammatory cells, including m acrophages and dendritic cells. Expression of IDO dampens the immune responses [ Brown 1991; Huang 2002; Mellor 2005 ; Schroecksnadel 2005 ]. Upregulation of IDO in infla mmatory cells has been shown to provide potent immunosuppression in auto immune disease, allergies, chronic inflammatory conditions, and chronic inf ection. Relevant to this protocol, IDO expression is also increased in macrophages and dendritic cells of melanoma metastases. Moreover, enhanced IDO expression in immune cells in the se ntinel node is an independent prognostic factor [ Speeckaert 2012 ]. IDO is also overexpressed by some cancer cel ls. Such overexpression leads to tryptophan depletion and kynurenine excess, leading to T-cell immunosuppression, which can prevent immune recognition and attack on malignant cells, and might afford a tumor-derived mechanism of immune escape [ Liu 2009]. Because of this, IDO expression and enzymatic activity corre lates strongly with prognosis in multiple cancers. In particular, IDO1 has been observed to be chronically activated and overexpressed in many cancers, including ovarian, colorectal, pancreatic, and prostate cancers, and melanoma. IDO expression and enzymatic activity correlates strongly with prognosis in multiple cancers [Liu 2009]. The expression of IDO1 correlates w ith more extensive disease and is an independent prognostic factor for reduced overall survival. In one study, moderate to strong cytoplasmic IDO expression was present in all (15/15) melanoma lymph node metastases in patients with poor survival [ Brody 2009]. In other tumor types, increased serum L-kynurenine concentrations predict a poor prognosis in patients with diffuse large B-cell lym phoma treated with R-CHOP (rituximab– cyclophosphamide, doxorubicin, vincristin e, and prednisone) chemotherapy [ Yoshikawa 2010]. In addition, increased tryptophan degr adation and increased IDO activity led to increased Kyn/Trp ratio in patients with ova rian cancer when compared with healthy controls. Furthermore, higher st ages of ovarian carcinoma were associated with a higher rate of tryptophan degradation, and K yn/Trp ratios correlated strongly with concentrations of cytokine interleukin 6 (IL-6), soluble IL-2 receptor alpha, tumor NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 10 necrosis factor alpha (TNF-alpha) recepto r, and the macrophage marker, neopterin [Schroecksnadel 2005]. Increased serum Kyn/Trp ratios have also been shown to correlate with immunological effects and disease progression in lung cancer and other cancers [ Liu 2009]. For example, patients with lung cancer had significantly higher Kyn levels, lower Trp serum levels, and elevated Kyn/Trp ratio compared to normal controls (Table 2.3) [Suzuki 2010]. Although substantial overlap be tween normal and lung cancer values was demonstrated, patients with more advanced cancer te nded to have higher abnormal values. Table 2.3 . Kyn and Trp Levels and Ratios in Patien ts with Lung Cancer vs Normal Controls Variable Normal controls Lung cancer P Kyn serum levels 2.30±0.56 µM 2.82±1.17 µM 0.0036 Trp serum levels 71.1±11.8 µM 62.6±15.8 µM 0.0007 Kyn/Trp ratio 32.9±9 47.1±21.3 <0.0001 Abbreviations: Kyn, kynurenine; Trp, tryptophan. 2.3.1 INCB024360 The novel small-molecule inhibitor of I DO1, INCB024360, is the first IDO1 selective inhibitor to undergo assessment in a human clinical trial. INCB024360 is specific for IDO1 with potency in the nanomolar range (IC 50=7.1–12.7 nM) in both human tumor cells and dendritic cells, and reduces trypt ophan-to-kynurenine conversion as assessed by biochemical and cellular assays [ Koblish 2010; Liu 2010 ]. It is highly potent in reversing IDO1-mediated suppression of T-cell proliferation in a dose-dependent manner (EC 50=17.7 nM) with potency cons istent with its inhibiti on of tryptophan-to-kynurenine conversion. It demonstrates high selectivity and does not inhibit other tryptophan- catabolizing proteins such as tryptophan 2,3-dioxygenase (TDO) or IDO2, nor does it influence tryptophan transporters. It has a greater than 1000-fold selectivity against a number of kinases, ion cha nnels, receptors, and transpor ters, and does not significantly induce or inhibit cytochrome P450 enzyme s. INCB024360 has an excellent preclinical pharmacokinetic profile with an oral bioavailability of 50% in rat and dog, with good tolerability and safety profiles in preclinical rodent and nonrodent studies of 28-day duration. 2.3.1.1 Animal Models In preclinical murine models, INCB024360 i nhibited IDO1 systemically in tumors and in tumor-draining lymph nodes with associated reduced tumor growth and enhanced lymphocyte activity. In mice, plasma Kyn levels decreased within 1 hour of oral INCB024360 administration and remained at minimal levels for as long as plasma levels of INCB024360 exceeded 1.2 µM (corresponding to the cellular IC 90 value adjusted for plasma protein bindi ng [~8 hours]). Consistent with specific inhibition of IDO1 by INCB024360, no reduction in plasma Kyn levels was observed in IDO1–genetically deficient mice, despite similar compound exposures. Furthermore, during maximal suppressi on of IDO1 by INCB024360 in wild-type NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 11 mice, the plasma Kyn levels were similar to the baseline plasma levels in IDO1- deficient mice, demonstrating that oral administration of INCB024360 results in efficient and selective IDO1 inhibition in vivo. Reduced levels of regulatory T cells (Tre gs) were demonstrated in the syngeneic tumors in mice that were administered INCB024360, compared with tumors in control mice. As a single agent, INCB024360 inhibited the growth of CT26 colon tumors, which express high levels of endogenous IDO1 when grown in syngeneic BALB/c mice [ Koblish 2010]. No effect was observed in tumor models using immunocompromised BALB/c nu/nu mice. Furt hermore, the effect of chemotherapy appeared to be enhanced with INCB024360 in preclinical models: in CT26 tumor– bearing mice, tumor growth was reduced by 33% when doxorubicin was administered alone, by 25% when INCB024360 was admi nistered alone, and by 66% when administered in combination. 2.3.1.2 Clinical Study INCB024360-101 INCB024360 is currently being evaluate d in an ongoing Phase I study (INCB 24360- 101) conducted by Incyte Corporation in s ubjects with refractory solid tumors [ClinicalTrials.gov Identifier: NCT 01195311]. The study has used a 3+3 dose escalation design, with doses of INCB024360 ranging from 50 mg once daily (qd) to 700 mg twice daily (bid). A total of 52 s ubjects have been treated: 3 initiated treatment at 50 mg qd, 4 at 50 mg bid, 3 at 100 mg bid, 6 at 300 mg bid, 11 at 400 mg bid, 5 at 500 mg bid, and 4 at 700 mg bid. In the food effect expansion cohort, 14 subjects were treated at 600 mg bid. INCB024360 has generally been well tole rated. The most common adverse events (AEs) (≥ 20%) include fatigue, nausea, decreas ed appetite, vomiting, constipation, abdominal pain, diarrhea, dyspnea, back pain, and cough. The most common grade 3 or 4 AEs were abdominal pain, hypokalemia, and fatigue (9.6% each). Despite 1 patient reporting a dose-limiting toxicity (DLT) of radiation pneumonitis at 300 mg bid, additional subjects were enrolled at this dose and no additional DLTs were reported. At 400 mg bid, 1 patient report ed a DLT of grade 3 fatigue, with no additional DLTs at this dose level. Another patient at 400 mg bid developed asymptomatic and transient hypopituitarism af ter 1 cycle of therapy and was able to continue dosing with INCB024360. Among the 18 subjects treated at either 600 or 700 mg bid, no DLTs were observed. Do se escalation was completed based on preliminary pharmacokinetic and pharmac odynamic data, which demonstrated that doses of INCB024360 above 300 mg bid achieved exposures exceeding the IC 90 determined in nonclinical models and, in pharmacodynamic assays, reached a plateau for Kyn/Trp levels and ratios. In the fina l cohort of 600 mg bid, the effect of food on INCB024360 absorption was evaluated. No objective responses were reported. At 2 months, stable disease was s een in 15 patients and lasted ≥4 months in 7 patients. The main AE reported in subjects receiving more than 2 cycles of INCB024360 has been grade 1 or 2 fatigue. NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 12 2.3.1.3 Clinical Study (INCB024360-201) INCB 24360-201 is a Phase Ib/II study of INCB024360 in combination with ipilimumab in subjects with advanced melanoma. Between April and August 2012, 8 subjects were enrolled in the Phase Ib portion of the study and were treated with INCB024360 300 mg bid (Cohort 1), 300 mg bid reduced to 100 mg bid (a continuation of Cohort 1 after hepatotoxity was identifie d), or 100 mg bid (Cohort 2), each in combination with 3 mg/kg of intr avenous (IV) ipilimumab every 3 weeks. The initial 3 subjects treated with 30 0 mg bid of INCB024360 plus ipilimumab developed grade 3 or 4 elevations of aspartate aminotransferase/alanine aminotransferase (ALT/AST) on study da ys 76, 41, and 44, respectively. None of these subjects had elevated bilirubin, liver failure, or other evidence of severe liver injury. These subjects were discontinue d from therapy and treated with IV methylprednisolone followed by oral prednisone on a tapering schedule until normal liver function recovered. At the time these elevations were reported, 4 additional subjects were already enrolled a nd receiving INCB024360 at 300 mg bid with 3 mg/kg IV of ipilimumab. All 4 of these s ubjects were evaluated and were found to have normal liver function tests (LFTs). Yet, in response to the LFT elevations in the first 3 subjects on study and after di scussions with the US Food and Drug Administration (FDA), the dose of INCB024360 was reduced to 100 mg bid in these 4 subjects, and screening and enrollment c ontinued to evaluate an initial dose of INCB024360 of 100 mg bid. After approximately 2–3 weeks of recei ving the reduced dose of INCB024360, 2 of the 4 subjects who had a dose reduction de veloped hepatic toxicity, including 1 subject symptomatic with abdominal discomfort and reported to have grade 4 elevation of ALT and AST with grade 1 elevation of bilirubin and normal coagulation parameters. These 2 subjects improved after treatment with IV methylprednisolone and oral prednisone, until liver functi on parameters normalized. All 4 subjects received INCB024360 at 300 mg bid for 10, 13, 14, and 52 days, respectively, before having their dose reduced to 100 mg bid for 16–18 days. Among the 4 subjects who had their dose reduced, the 2 subjects who developed hepatic toxicity received INCB024360 at 300 mg bid for 13 and 14 da ys, respectively. The duration of exposure to INCB024360 at 300 mg bid was not clearly related to the risk of developing hepatic toxicity, since the subjec t with the longest exposure (52 days) did not develop hepatic toxicity after th e dose reduction. Thus, a dose-response relationship might exist for the de velopment of hepatic toxicity. After these additional reports of hepatic toxicity, all subjects were removed from study treatment, and screening for the study was suspended. An additional subject had been enrolled in Cohort 2 and received a single dose of INCB024360 at 100 mg at the time the decision was made to stop dosing INCB024360 in all subjects and to suspend enrollment into the study. This subject has continued to receive ipilimumab as part of standard treatment for advanced melanoma, with no hepatic toxicity reported. Among the 7 subjects in Cohort 1, 1 completed a ll 4 of the planned ipilimumab infusions while in the study. Three of the remaini ng 6 subjects have gone on to receive NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 13 additional doses of ipilimumab, including 1 who had developed hepatic toxicity from the combination of INCB024360 and ipilimuma b. No additional hepatic toxicity has been reported in these subjects. The st udy was recently reopened and is evaluating lower doses of INCB024360 (25–100 mg bid) in combination with ipilimumab. Of note, in the Phase I study (INCB 24360-101) , 7 subjects were enrolled with melanoma, including 4 with prior ipilimumab therapy. None of these subjects were reported to have related grade 3 or 4 elevations in LFTs. Plasma levels of INCB024360 and plasma le vels of Kyn were measured in serial samples. Steady-state linear pharmacokinetics were obtained on or before day 8, and appreciable drug accumulation with increase s in mean area under the curve (AUC) by 30% between analysis on day 1 and 15 wa s evident. The half-life was 2.3–4 hours. Pharmacodynamic correlates suggest that signi ficant target inhibi tion was achieved at bid doses ≥ 300 mg bid, resulting in steady-st ate exposures that exceeded the IC 90. As a pharmacodynamic effect, blood samples fo r tryptophan and kynurenine levels show significant target inhibi tion with doses of 50 mg bid or greater. In patients showing elevations at baseline, ≥300 mg bid doses normalize kynurenine levels. IHC staining of 11 patients demonstrated that IDO1 tumor expression could be observed in tumor cells, infiltrating inflammatory cells, or both, in 10 out of 11 samples, and CD3+ T cells and Foxp3+ Tregs were present in all examined samples. Based on the tolerability and thes e pharmacokinetic and pharmacodynamic observations, a dose of 600 mg bid has been determined as the recommended Phase-II dose as monotherapy. In recent preliminary results from a Phase ½ study of INCB024360 combined with ipilimumab in patients with advanced melanoma data show ed that in immunotherapy- naïve patients given 25 mg BID or 50 mg BID, 42% of patients demonstrated an objective response and 75% of patients de monstrated disease control by irRC. [Gibney; Hamid 2014 ] It is therefore proposed that reducing the dose of INCB024360 from 600 mg BID to 300 mg BID in all patients may help prevent adverse events and dose limiting toxicities without limiting biological effect. In unpublished preliminary data from Incy te, kynurenine concen trations and the Kyn/Trp ratio were higher in the serum of most patients compared to controls and decreased to levels below the median for nor mal, after treatment with inhibitor when treated with doses that exceeded the IC 90 (i.e., >300 mg bid [N=19 patients]) (Table 2.4). Table 2.4 . Changes in Kynurenine Concentrations and Kyn/Trp Ratios Serum Variables Control Patients median Cancer Patients median (range) Post-inhibitor median (range) All patients (N=19) Kynurenine, µM 2.0 2.1 (1.3–4.2) 0.97 (0.5–1.7) Kyn/Trp ratio, nmol/µmol 29.9 37.4 (17.8–68.9) 18.3 (N/A) Select patients* Kynurenine, µM 2.0 2.7 (2.1–4.2) 1.1 (0.5–1.7) Kyn/Trp ratio, nmol/µmol 29.9 42.0 (34–69) 18.6 (8–43) NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 14 * with Kyn levels >2 µM (n=8) or Kyn/Tyr ratio >30 nmol/µmol (n=14) Abbreviations: Kyn, kynurenine; N/A, not available; Trp, tryptophan. 2.3.2 MELITAC 12.1 Melanoma Vaccine MELITAC 12.1 consists of 12 melanoma-asso ciated peptides from melanocytic differentiation proteins (tyrosinase and gp100) and cancer testis antigens (MAGE-A1, MAGE-A3, MAGE-A10, and NY-ESO-1), pl us a tetanus peptide that binds promiscuously to HLA-DR molecules and stimulates Th1 dominant helper T cell responses (nonspecific tetanus helper peptide) [ Slingluff 2001 ; Slingluff 2007 ]. In a Phase II clinical trial of adjuvant therapy in patients with resected stage IIB to IV melanoma, MELITAC 12.1 was fo und to be more efficacious (i.e., immune response was higher) and demonstrated more complete immu nologic responses (i.e., greater cumulative T-cell responses) than vaccines with fewer peptides [ Slingluff 2007 ]. The 12-peptide vaccine was found to induce substantially higher immune response rates: 100% of vaccinated patients had immune responses to at least 1 peptide (detectab le in the blood in 83% or in the sentinel immunized lymph nodes in 92%). The immunogenicity of individual peptides was maintained despit e competition with additional peptides for binding to major histocompatibility complex (M HC) molecules, with a broader and more robust immune response. Clinical outcome with this peptide vaccine correlated with the immune responses measured in peripheral blood lymphocytes. In subsequent trials, Mel43 and Mel44, when administering the vaccine without granulocyte-macrophage colony-stimulating factor (GM-CSF), imm unogenicity was significantly enhanced. In earlier trials with GM-CSF, immune responses were detected after in vitro sensitization. In subsequent trials without GM-CSF, immune responses could be detected directly ex vivo in 70–73% of patients [ Slingluff 2011 ; Slingluff 2009 ]. This vaccine was well tolerated. In the most recent Mel44 trial (n=170), 8% of patients reported unexpected grade 3 treatment -related AEs, plus 10% with grade 3 injection-site ulceration and 7% with grade 3 injection-site induration; the injection-site reactions were expected and not dose-limiting. One grade 4 to xicity was reported (hypoglycemia, <1%). Other common toxicities were evident for approximately 24 hours and commonly included fatigue, dyspnea, headache, and myalgi a. Mild AEs of rigor s/chills, anorexia, nausea, sweating, fever, and flushi ng were commonly experienced [ Slingluff 2011 ]. No concurrent comparator for efficacy was established in the nonrandomized trial of MELITAC 12.1. However, the disease-free surviv al (DFS) data with this multipeptide vaccine compared favorably to adjuvant tr ials of high-dose IFN (E1694 clinical trial), favorably with respect to studies E1684 and E1690, and analogously to the Canvaxin trial in patients with resected stage III melanom a. The median DFS was 35 months (2.9 years) with a corresponding 1-year DFS of 76% (95% confidence interval [CI], 60–86%), 2-year DFS of 59% (95% CI, 42–72%), and 3-year DFS of 47% (95% CI, 27–64%). In the more recent Mel43 and Mel44 trials, 3-year DFS was 52% in both trials (95% CI, 43–61%) and overall survival at 3 years was 76% (95% CI, 67–83%) and 79% (95% CI, 71–86%) respectively [ Slingluff 2009 ; Slingluff 2011 ]. NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 15 This and similar multipeptide vaccine preparations have been evaluated in prospective randomized Phase II trials with several lo cal adjuvants (e.g., GM-CSF, dendritic cells, incomplete Freund’s adjuvant [IFA]), systemic low-dose IL-2, and systemic cyclophosphamide [ Slingluff 2004 ; Slingluff 2007 ; Slingluff 2009 ; Slingluff 2011 ]. Vaccination in IFA was more immunogenic th an pulsing peptides on dendritic cells, a finding supported by a separate randomized trial [ Slingluff 2003 ; O’Neill 2009 ]. No immunologic or clinical bene fit was evident by adding lo w-dose IL-2, GM-CSF, or cyclophosphamide [ Slingluff 2004 ; Slingluff 2009 ; Slingluff 2011 ]. Thus, the formulation proposed for this trial is peptide in IFA. 2.4 Rationale The central thesis of this prot ocol is that an inhibitor of IDO1 may decrease melanoma- associated immune suppression and immune cell–associated immune suppression and increase the immune response at sites of tumor, specifica lly increasing both the total number of T cells and the number of melanoma antigen–specific T cells infiltrating the tumor. IDO expression and function arises from 2 potential sources: (1) tumors can overexpress IDO and (2) inflammatory cells can upregulate IDO in response to the IFN- gamma secreted by activated T lymphocyt es. Overexpressed and upregulated IDO subsequently depletes tryptophan and in creases tryptophan catabolites, including kynurenine, that cause antigen-presenting ce lls (APCs) and cytotoxic T cells to be ineffective against established tumors. Data from animal experiments [ Munn 2012] demonstrate that IDO induction after vaccination serves as a negative feedback loop that dampens the immune response to dendritic ce ll–based cancer vaccines. An effective IDO inhibitor may reverse this particular form of tumor-associated immune suppression and increase the response to tumor vaccines by augmenting T-cell responses to cancer antigens. A relatively nontoxic regimen of INCB024360 as monotherapy has been shown to normalize serum Kyn/Trp ratios in patient s with cancer. That same regimen is hypothesized to inhibit the effect of IDO expression at sites of tumor deposition and thereby unleash the nascent or vaccine-induced antitumor immune response either at sites of tumor deposition or in the tumor-draining lymph nodes. In addition, the same regimen is hypothesized to increase the systemic i mmune response to cancer vaccines and/or allow vaccine-induced T cells to replicate and to function better at sites of tumor deposition. Because the melanoma multiple peptide vaccine, MELITAC 12.1, stimulates immune responses, has an excellent safety profile, and is presumed efficacious in the adjuvant setting, treating patients with INCB024360 before vaccination with MELITAC 12.1 might increase the immune response to antigens expressed by melanoma cells and allow for an increased antitumor immune response. 2.4.1 IDO Inhibition in Melanoma and T-cell Infiltration into Tumor IDO is strongly associated with poor prognosis [ Gajewski 2005 ; Brody 2009], and dendritic cells in lymph nodes dr aining melanomas express IDO [ Lee 2003 ]. High expression of IDO in the sentinel nodes of pa tients with melanoma is strongly associated NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 16 with increased tumor burden and shorter survival [Lee 2011 ; Speeckaert 2012 ]. On the other hand, a recent biomarker study found that high IDO expression in tumors before treatment was associated with improved outcome after therapy with ipilimumab, and that increases in T-cell infiltration during treatm ent were also associated with improved clinical outcome [ Hamid 2011]. IDO is induced by IFN-gamma produced by T ce lls, and thus represents a potent immune regulatory pathway induced when activ ated T cells infiltrate tumors [ Mackler 2003 ; Zhang 2011; Zhao 2012]. The findings from this recent study emphasize the highly complex interplay of immune activation and suppression in the melanoma microenvironment and its association with T-ce ll infiltration. They suggest that patients whose tumors express high levels of IDO are those with immune signatures for whom an immune therapy may have a higher chance of success, and that clinical benefit is associated with an increase in T-cell infiltration of melanoma metastases. 2.4.2 Lymphocyte Infiltrates Patients with brisk lymphocytic infiltrates in primary melanomas have lower risk of metastasis and death than patients with sparse or absent infiltrate [ Erdag 2012]. In addition, Slingluff has established that lymphoc ytic infiltrate in metastatic melanomas correlates with increased survival. Slinglu ff evaluated the composition and organization of immune cells that infiltrated 183 melanom a metastases in humans. CD45+ cell counts ranged from 0 to 2,142 per mm2 (Table 2.4.2). The most preval ent immune cells in the first metastases were T cells (53%), of which CD8+ T cells predominated (35%) and CD4+ T cells were less common (19%). B cel ls (CD20) and plasma cells (CD138) were present at high numbers (25% and 8%, respectively). Macr ophage lineage cells (CD163) accounted for 13% of the infiltrates. NK ce lls (CD56) and mature dendritic cells (DC- LAMP) were rare across all samples, with mean proportions of less than 1% each. Table 2.4.2. Distribution of Immune Ce ll Subsets Among 183 Metastases Studied [Erdag 2012] Subset N Mean Min 25% Median 75% Max CD45 183 246 0 31 117 343 2142 CD138 183 26 0 0 2 23 488 CD20 182 65 0 0 5 47 1796 CD3 183 153 0 20 83 226 1049 CD4 183 52 0 3 16 60 602 CD8 182 103 0 15 71 153 462 CD56 183 3 0 0 0 2 85 PD1 183 18 0 0 2 20 262 Foxp3 182 19 0 0 8 23 175 CD163 182 39 0 2 18 53 360 DC-LAMP 1 0 0 0 1 19 Three histologic patterns of immune cell in filtration were identified. Immunotype A was characterized by no immune cell infiltrate. Immunotype B was ch aracterized by infiltration of immune cells limited only to regions proximal to intratumoral blood vessels. Immunotype C was characterized by a diffuse immune cell infi ltrate throughout a metastatic tumor. These NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 17 immunotypes represented 29%, 63%, and 8% of metastases with estimated median survival periods of 15, 23, and 130 months, respectively. The best correlates with survival were with CD8+ T cells. Whereas higher densities of CD8+ T cells correlated best with survival, a higher density of CD45+ leukocytes, T cells, and B cells also correlated with in creased survival. These reveal how the immune microenvironment can a ffect outcomes and also provide a method to quantify the effect of the IDO1 i nhibitor on immune cell infiltration. 2.4.3 Data on combination effects of checkpoint blockade antibodies and IDO inhibition. The success of checkpoint blockade ther apy for melanoma has transformed the management of patients with advanced me lanoma, but most patients treated with antibodies to CTLA4 or PD-1 will progress de spite those therapies. Combined therapy with antibodies to CTLA4 and PD-1 have induced higher response rates, and durable clinical benefit, but at the price of increased toxicity. There is a need for additional combination therapies that may enhance clinical benefit, ideally at lower toxicity than combination of CTLA4 and PD-1 antibodies. IDO and PD-1 can mediate immune escape of melanoma. As effector T cells inf iltrate melanoma metastases, they produce interferon-gamma, which in turn can induce both PD-L1 and IDO. Blockade of PD-1/PD-L1 can further increase T cell infiltration; so there is rationale to expect that IDO may mediate progression of can cer on PD-1 antibody therapy. [ Spranger 2015 ], [Gajewski 2015 patent ] Combination of ipilimumab and the IDO inhibitor INCB024360 has induced significant transaminase elevations in 5 of 7 patien ts, when INCB024360 was administered at 300 mg bid; the study was continued in 8 patie nts at a much lower dose of INCB024360 (25 mg bid), with disease control observed in 6 of those 8 patients and irPR in 3 (38%). Thus, this small series suggests en couraging clinical ou tcomes with that combination, but there was unacceptable toxicity with a higher dos e of INCB024360. Treatment of patients with another IDO inhibitor (D-1MT) af ter prior ipilimumab also wa s associated with dramatic toxicities, including hypophysitis, also raising questions about the toxicity of CTLA-4 blockade plus IDO inhibition. PD-1 blockade with pembrolizumab or nivolum ab has become standard first-line therapy for advanced melanoma (based on data reporte d in 2015); yet most patients treated with these agents will progress on therapy. Addition of IDO inhibition to these patients offers promise for improving clinical response rate s, thus, a cohort has been added to allow addition of INCB024360 early after the last dose of PD-1 antibody (o r PD-L1 antibody). If data from this cohort support safety a nd clinical activity of INCB024360 when added to patients progressing on PD-1 antibody, thes e data will support future study of adding INCB024360 while continuing PD-1 antibody af ter failure to respond. Also, biopsies obtained after progression on PD-1 antibody and after adding INCB024360 will provide insight into the role of IDO in mediating fail ure of PD-1 inhibition and the changes in the tumor microenvironment of adding IDO blocka de while PD-1 blockade remains active. NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 18 2.4.4 Correlative Studies To meet the primary objectives, tumors will be biopsied before therapy, after 3 weeks of INCB024360 treatment, and 1 week after th e third vaccine plus INCB024360. Because immunotype and immunohistologic characteristics of tumor- infiltrating immune cells are associated with clinical outcome in metastatic melanoma [Erdag 2012], tumors will be assessed by IHC for T-cell infiltration patterns and infiltration with cells expressing CD8. CD8+ T cell infiltration will be reported as cells per mm2 of cross-sectio nal tumor area. Note: Optimally, patients will have tumor approachable for three serial biopsies during the trial. For patients with only one or two tumors approachable for biopsy, available tumor blocks from prior biopsies (without inte rvening systemic treatment) can serve as the pretreatment sample. The biopsy after 3 weeks of INCB024360 will be considered essential. Biopsy 1 week after the thir d vaccine plus INCB024360 will be considered optional. To characterize the immune cell infiltrate furt her, the tumors will also be assayed for a variety of antigens including CD4, CD8, CD56, CD34, CD45, CD20, CD3, CD8, CD138, CD163, DC-LAMP, FoxP3, and PD-1. IHC staining of tumor biopsies will also be performed to detect IDO1 expression, and th e presence and pattern s of CD8+ and CD4+ T cell infiltration, Foxp3 protein, and FoxP3 Tr eg infiltration. Gene signature will be assessed using an Affymetrix chip with focus on IFN signaling, IDO expression, and tumor rejection [ Weiss 2011 ] with each patient serving as their own control. Gene signatures on the tumor will be determined by Ena Wang, PhD, and Franco Marincola MD, Chief, Infectious Disease and Imm unogenetics Section in the Department of Transfusion Medicine, Clinical Center of the National Institutes of Health. A number of other (secondary) correlative stud ies will be conducted to evaluate whether a regimen of INCB024360 that normalizes serum Kyn/Trp ratios plus MELITAC 12.1 vaccine changes the level or character of the vaccine-induced CD8+ and CD4+ T-cell immune responses as measured in peripheral blood, compared to each patient’s baseline. Evaluations will occur at baseline (week 0), again at week 3, week 6, some are done at week 14 and they are repeated optionally at week 16 only if the opti onal biopsy is done at week 16. Effector 8+ T-cell responses to MELITAC 12.1 will be measured by IFN- gamma enzyme-linked immunosorbent spot assay (ELISpot) and with tetramers/multimers to 8 of the peptides in the vaccine. Effector CD4+ T-cell responses to MELITAC 12.1 will also be tested. Assays will be performed in the CITN Central Laboratory of the Tumor Vaccine Group led by No ra Disis, MD, in collaboration with the immune monitoring laboratory of Craig Slingluff, MD. The extent to which INCB024360 plus MELI TAC 12.1 vaccine alters the number and character of PBMC (including T and NK cells as evaluated by multiparameter flow cytometry) will be calculated by assessing the effect on T lymphocyte number and phenotype (i.e., CD8+, effector memory CD 8+, central memory CD8+, CD4+, CD4+ Treg, naïve subsets). The frequency and percen tage of PBMC subsets will be determined using multiparameter flow cytometric analysis on whole blood. The markers include NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 19 CD3, CD8, CD4, CD56, Foxp3, CD127, CD45RA, CD45RO, CCR7, CD28, CD27, CD25, CD122, CD86, CD14, CD16, CD19, CD123, and CD11c. Changes in myeloid-derived suppressor cell populations will also be assessed by flow cytometry. Assays will be performed in the CITN Central Laboratory of the Tumor Vaccine Group led by Nora Disis, MD, in collaboration w ith the immune monitoring labora tory of Craig Slingluff, MD. 3. PATIENT SELECTION 3.1 Eligibility Criteria 3.1.1 Patients must have malignant melanoma validated by histology or cytology. Patients may have had primary cutaneous, mucosal, or oc ular melanoma or metastasis from an unknown primary site. NOTE: Patients must have measurable disease, defined as at least 1 lesion that can be accurately measured in at least 1 dimensi on (longest diameter to be recorded for non- nodal lesions and short axis for nodal lesions) as ≥20 mm with conventional chest x-ray or as ≥10 mm with spiral computed tomography (CT) scan, magnetic resonance imaging (MRI), or calipers by clinical exam. See Section 11 for the evaluation of measurable disease. 3.1.2 Unresectable stage III or IV melanoma validated by clinical criteria (including recurrent melanoma), or patients with multiple skin/sof t tissue metastases of melanoma that may be resectable but are judged to have a future recurrence risk exceeding 70% (e.g., large adenopathy, distant skin metastases or multiple in-transit melanoma metastases). Tumor deemed amenable to biopsy (excisional, incisional, or core, with at least 100 mm 3 tumor volume per biopsy date) and fine-n eedle aspiration (FNA) biopsy. Note : Optimally, patients will have tumor approachable for three serial biopsies during the trial. For patients with only one or two tumors approachable for biopsy, available tumor blocks from prior biopsies can serve as the pretreatment sample, but only if formalin-fixed tumor tissue is available and adequate to provide at least 20 unstained slides with sufficient tumor for analysis. NOTE: Patients with unresectable advanced stage III or IV melanoma (including recurrent melanoma) are only eligible if they have failed at least one other first-line systemic therapy (other than adjuvant thera py). Exceptions to this requirement are those patients who have refused and/or are inel igible for other systemic therapies. NOTE: BRAFi should be considered for all ‘unr esectable” or metastatic melanoma with BRAFV600E mutation; for low burden in-transit disease patients may enter trial without prior systemic therapy. 3.1.2.1 Stage IV no evidence of disease (N ED) is excluded by this criterion NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 20 3.1.3 Prior and concurrent therapy 3.1.3.1 Patients may have had prior systemic th erapy without constr aint on the number of prior treatment regimens except: a) Patients may not have had >450 mg/m2 doxorubicin b) Patients may not have had >3000 cG y to fields encompassing the entire pelvis 3.1.3.2 Patients must not be on any other sy stemic therapy within the following intervals before study enrollment: a) 1 week after stereotactic radiosur gery of the brain or comparable technology b) 4 weeks after cytotoxic chemotherapy or external beam radiation therapy c) 6 weeks after chemotherapy regimens including BCNU (carmustine) or mitomycin C d) patients who experience melanoma pr ogression (by RECIST 1.1 criteria) while on or after treatment with PD-1 or PDL-1 antibody may enroll on this study. NOTE: Patients must be off PD-1/PDL- antibody for at least 2 weeks to assess for delayed toxicity before be ing enrolled and receiving INCB024360. Patients who are enrolled 2 weeks and up to 6 weeks after the last dose of PD- /PDL-1 antibody will enroll in c ohort B and receive 100mg BID of INCB024360. Patients enrolled beyond the 6 week period after failing anti-PD-1/PDL-1 will be enrolled in cohort A. Cohort A patients will receive 300mg BID of INCB024360. Patients must not have active grade 2 auto immune toxicities attributed to these antibodies at study entry. e) 8 weeks after ipilimumab, othe r CTLA-4 antibody or other immunologically active antibody. NOTE: Patients receiving prior CTLA-4, anti-PD1 antibody or other immunologic therapy must show eviden ce of normal pituitary function at baseline and must not have active grade 2 autoimmune toxicities attributed to these antibodies at study entry. 3.1.4 Age ≥18 years [NOTE: Because insufficient dosing AE data are currently available on the use of INCB024360 in combination with MELITAC 12.1 in patients <18 years of age, children are excluded from this study, but will be eligible for future pediatric trials.] 3.1.5 ECOG performance status 0–1 (Karnofsky ≥ 70%, see Appendix A ). NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 21 3.1.6 Life expectancy of at least 6 months 3.1.7 Patients must have normal organ and marrow function as defined below:  leukocytes ≥3,000/mcL  absolute neutrophil count ≥1,500/mcL  platelets ≥75,000/mcL  hemoglobin >9g/dL  total bilirubin <1.5 × instit utional upper limit of normal [bilirubin <3 × institutio nal upper limit of normal for Gilbert’s syndrome]  AST(SGOT)/ALT(SGPT) Up to 2.5 times ULN  creatinine <1.5 × institutional upper limit of normal OR  creatinine clearance ≥60 mL/min/1.73 m2 for patients with creatinine levels above institutional normal.  PT, INR ≤1.5 × institutional UL N unless patient is therapeutically anticoagulated. If on anticoagulants, PT/INR need to be within appropriate anticoagulation limits for the clinical indication. Patients who are receiving anticoagulants may participate in the trial if their anticoagulation can be stopped safely for several days at the time of each biopsy.  TSH, Up to 4 times ULN if T4 is normal  T4 Within normal limits. If abnormal and patient is receiving thyroid replacement therapy, the thyroid medication may be adjusted and the T4 may be re-tested. 3.1.8 Patients must express HLA-A1+, -A 2+, or -A3+ (80% of patients) 3.1.9 LDH <5 × upper limits of normal [NOTE: These criteria will select against patients with bulky disease and will select for patients with less disease and earlier disease.] 3.1.10 Participants must not have had prior autoimmune diso rders requiring cytotoxic or immunosuppressive therapy, or autoimmune di sorders with visceral involvement. Participants must not have an active or inactive autoimmune disorders (e.g., rheumatoid arthritis, moderate or severe psoriasis, multiple sclerosis, inflammatory bowel disease, etc.) Participants who are receiving therapy for an autoimmune or inflammatory disease requiring these therapies are also excluded. 3.1.11 The following will not be exclusionary:  Resolved ipilimumab associated inflammatory disease  The presence of laboratory evidence of au toimmune disease (e.g., positive antinuclear antibody [ANA] titer) without associated symptoms  Subjects with vitiligo, thyroidi tis, or atopic dermatitis, but otherwise not meeting this criterion may be enrolled. Individual cas es can be discussed with the sponsor. NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 22 3.1.12 Not likely curable with surgery alone 3.1.13 Not currently receiving therapy 3.1.14 Females of childbearing potential must have a negative pregnancy test within 48 hours before initiating protocol therapy. NOTE: The effects of INCB024360 and MELITA C 12.1 on the developing human fetus are unknown. For this reason, women of child-bearing potential and men must agree to use adequate contraception (hormonal or barr ier method of birth control or abstinence) before study entry and for the duration of study particip ation. Should a woman become pregnant or suspect she is pregnant while she or her partner is participating in this study, she should inform her treating physician im mediately. Men treated or enrolled on this protocol must also agree to use adequate contraception be fore the study, for the duration of study participation, and 4 months after completing INCB024360 and MELITAC 12.1 administration. NOTE: Subjects are considered not of child b earing potential if they are surgically sterile, have undergone a hysterectomy, b ilateral tubal liga tion, or bilateral oophorectomy, or are postmenopausal. Menopause is the age associated with complete cessation of menstrual cycles and menses, a nd implies the loss of reproductive potential. By a practical definition, th e term assumes menopause after 1 year without menses with an appropriate clinical prof ile at the appropriate age. 3.1.15 Ability to understand and willingness to si gn a written informed consent document 3.2 Exclusion Criteria 3.2.1 Patients who have had cytotoxic chemothe rapy, radiotherapy, IFN, immunosuppressive therapy, or steroids within 4 weeks (6 week s for nitrosoureas or mitomycin C) before entering the study or those who have not recove red from AEs due to agents administered more than 4 weeks earlier 3.2.2 Ipilimumab, or other immunol ogically active therapy within 8 weeks of enrollment. NOTE: Patients who experience melanoma progr ession (by RECIST 1.1 criteria) while on or after treatment with PD-1 or PDL-1 an tibody may enroll on this study (per section 3.1.3.2 above) 3.2.3 Active immunosuppressive therapy, including concurrent systemic immunosuppressive therapy or steroid therapy with more than 7 c onsecutive days of ster oids within the prior 4 weeks – The use of prednisone or equivalent <0.125 mg/kg/day (absolute maximum of 10 mg/day) as replacement therapy is permitted. – Inhaled corticosteroids are permitted. NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 23 3.2.4 Cardiovascular disease that meets one of th e following: congestiv e heart failure (New York Heart Association Class II I or IV), active angina pect oris, or recent myocardial infarction or acute coronary syndrome (within the last 6 months) 3.2.5 History of peripheral vascular disease (PVD) that has required surgical or percutaneous intervention or documented PVD that require s medical management with medications such as ASA + Clopidogrel. Patients with Diabetes that is not well controlled are excluded from participation. Not well controlled is defined as a Hgb A1C of greater than 7.5%. 3.2.6 Current or history of systemic autoimmune disease requiring syst emic therapy, including significant autoimmunity associated with prior ipilimumab therapy or therapy with antibodies to PD-1 or PD-L1 3.2.7 Cirrhosis, chronic hepatitis C virus positivity, or chronic hepatitis B infection. Subjects who may not tolerate immune-mediated hepatitis due to compromised hepatic reserve are also excluded from participation in cluding: 1) Subjects with extensive liver metastasis (as judged by the investigator) 2) Subjects who drink more than two standard alcoholic beverages per day on a regular ba sis 3) Subjects who consume more than 2 grams of Acetaminophen per da y on a regular basis. – A positive hepatitis B serology indicative of previous immunization (i.e., HBsAb-positive and HBcAb-negative), or a fully resolv ed acute hepatitis B infection is not an exclusion criterion. 3.2.8 Patients who are receiving any other investigational agents for melanoma 3.2.9 Patients who have had a grade one or grade tw o gastrointestinal a dverse event during or after receiving anti-CTLA-4, anti-PD1 or an ti-PD, without a colonoscopy verifying complete resolution of the adverse event. 3.2.10 Patients who have experienced bowel perfor ation, neurologic involvement, Guillain Barré syndrome, Myasthenia Gravis, Steven Johnson syndrome and other intractable events or grade 4 non-laboratory toxicity. 3.2.11 Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstabl e angina pectoris, cardiac arrhythmia, or psychiatric illness/social situ ations that would limit comp liance with study requirements 3.2.12 Pregnancy, nursing, or unwilling to take ad equate birth control during therapy NOTE: Pregnant women are excluded from this study because INCB024360 and MELITAC 12.1 have the potential for teratogenic or abortifacient effects. Because of unknown but potential risks for AEs in nursing infants secondary to treatment of the mother with INCB024360 and MELITAC 12.1, br eastfeeding should be discontinued if the mother is treated with INCB024360 and MELITAC 12.1 These potential risks may NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 24 also apply to other agen ts used in this study. 3.2.13 Known HIV or other history of immunodeficiency disorder NOTE: HIV-positive patients taking combination antiretroviral therapy are ineligible because of the potential for pharmacoki netic interactions with INCB024360 and MELITAC 12.1 . In addition, these patients are at increa sed risk of lethal infections when treated with marrow-suppressive therapy. A ppropriate studies will be undertaken in patients receiving combination anti retroviral therapy when indicated. 3.2.14 Extensive active brain disease, including sympto matic brain metastases or the presence of leptomeningeal disease o Patients with brain metastasis, after defin itive therapy with surgery or stereotactic radiation and stable off steroids for >4 weeks, are eligible. 3.2.15 Any malignancy that has not been in complete remission for at least 3 years NOTE: Patients with cured basal or squamous cell skin cancer are not excluded. Patients with a history of excised in situ cancers, includi ng breast, cervical, colon, superficial bladder, prostate or other body system are not excluded. Study entry will be allowed at the di scretion of the Principal Investigator. NOTE: Recurrence of the in situ cancer or tumo r at the time of study entry would be exclusionary. 3.2.16 Monoamine oxidase (MAO) inhibitor use within the past 3 weeks or prior evidence of serotonin syndrome 3.2.17 History of allergic reactions attributed to compounds of similar chemical or biologic composition to INCB024360, MELITAC 12.1, or other vaccine components 3.2.18 Prior organ allograft or allogeneic transplantat ion, if the transplanted tissue is still in place 3.2.19 Medical or psychiatric illness that would, in the opinion of the investigator, preclude participation in the study or the ability of patients to provide informed consent for themselves 3.2.20 History of pulmonary disease such as emphysema or chronic obstructive pulmonary disease (COPD), (FEV1>60% of predicted for height and age). Pulmonary function tests (PFTs) are required in patients with pr olonged smoking history or symptoms of respiratory dysfunction. NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 25 3.2.21 Use of any UGT1A9 inhibitor including: acitretin, amitriptyline, androsterone, cyclosporine, dasatinib, diclofenac, diflunisal, efavirenz, erlotinib, estradiol (17-beta), flutamide, geftinib, gemfibrozil, glycyrrhetin ic acid, glycyrrhizin, imatinib, imipramine, ketoconazole, lineoleic acid, mefenami c acid, mycophenolic acid, niflumic acid, nilotinib, phenobarbital, phenylbutazone, phenytoin, probenecid propofol, quinidine, ritonavir, Sorafenib, sulfinpyr azone, valproic acid and vera pamil from screening through follow-up period. 3.2.22 Low-dose Coumadin (1 mg) is acceptable; however, doses that increase INR are not permitted. If an alternative to Coumadin-bas ed anticoagulants cannot be used, the INR should be monitored weekly after initiation of therapy and upon discontinuation of INCB024360, until INR normalization. 3.3 Inclusion of Women and Minorities Both men and women of all races and ethnic grou ps are eligible for this trial (Table 3.3).