Abstract A major challenge to further development of immune checkpoint therapies (ICT) for advanced cancer is the inherent toxicity of higher-order multidrug combinations likely needed to eradicate metastatic disease. Recently, early clinical studies have investigated a new general approach to address this challenge, termed cryo-immune vaccination (CIV). The CIV approach by SYNC-T therapy involves cycles of local cryolysis of a solid tumor followed by infusion of the same lesion with a low-dose multi-API formulation. CIV has never been explored methodically in preclinical or clinical settings, so the basis for systemic responses that have been observed in early clinical testing are not fully understood. Accordingly, as trials of the safety and efficacy of CIV proceed, there is now a need of preclinical model systems to help illuminate the mechanistic foundations of the approach, investigate safety and efficacy, and learn where and how it might be best leveraged. To develop the conceptual framework of CIV, we adapted the orthotopic metastatic 4T1 breast cancer model used extensively in our laboratory as a foundation to study CIV antitumor responses and mechanisms of action. Briefly, breast tumors were initiated in naïve murine subjects by orthotopic injection of 1x104 cells into the mammary fad pads of immunocompetent BALB/c hosts. When tumors reached ~5-7 mm, a time when occult pulmonary metastases have seeded, primary tumors were exposed surgically on skin flaps and contacted directly with a liquid nitrogen-cooled pointed steel rod for 15-30 sec. The rod was then withdrawn and tumor tissue allowed to thaw fully before injection into the cryolysed tumor of a selected ICT multidrug cocktail (or negative control murine IgG) in 25-50 µl total volume. The selected multidrug cocktail was modeled on SYNC-T combination therapy currently under study in a Phase 1 clinical trial (Syncromune, Inc.) Briefly, the ICT drug cocktails were variably composed of murine-reactive antibodies against CTLA4, PD-1 or OX40 (agonist), CD40L (agonist) and CpG oligonucleotide. As a starting point, allometric scaling was used to model dosing analogous to the proprietary ICT drug formulations SV-101 and SV-102 in clinical testing (Syncromune, Inc.). For exploratory toxicology, a standard panel of blood-borne liver and kidney toxicity biomarkers was assessed. For exploratory pharmacology, a ‘super mouse’ design was employed with ELISA-based determinations to measure antibody distribution in blood serum. To gain initial mechanistic insights, responses will be compared in host mice that are genetically deficient in selected components of the adaptive or innate immune systems. Progress in methodological and mechanistic studies will be presented. Citation Format: James B. DuHadaway, Alexander J. Muller, Lisa D. Laury-Kleintop, U. Margaretha Wallon, Susan K. Gilmour, Marie Webster, Jason R. Williams, Gabriela R. Rossi, Mario R. Mautino, Jonathan Lewis, Charles J. Link, George C. Prendergast. Cryo-immune vaccination (CIV) by SYNC-T therapy: Preclinical modeling of a novel device-multidrug immunotherapeutic approach to eradicate advanced metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB355.

BackgroundThe indoleamine 2,3-dioxygenase (IDO) pathway is a key counter-regulatory mechanism that, in cancer, is exploited by tumors to evade antitumor immunity. Indoximod is a small-molecule IDO pathway inhibitor that reverses the immunosuppressive effects of low tryptophan (Trp) and high kynurenine (Kyn) that result from IDO activity. In this study, indoximod was used in combination with a checkpoint inhibitor (CPI) pembrolizumab for the treatment for advanced melanoma.MethodsPatients with advanced melanoma were enrolled in a single-arm phase II clinical trial evaluating the addition of indoximod to standard of care CPI approved for melanoma. Investigators administered their choice of CPI including pembrolizumab (P), nivolumab (N), or ipilimumab (I). Indoximod was administered continuously (1200 mg orally two times per day), with concurrent CPI dosed per US Food and Drug Administration (FDA)-approved label.ResultsBetween July 2014 and July 2017, 131 patients were enrolled. (P) was used more frequently (n=114, 87%) per investigator’s choice. The efficacy evaluable population consisted of 89 patients from the phase II cohort with non-ocular melanoma who received indoximod combined with (P).The objective response rate (ORR) for the evaluable population was 51% with confirmed complete response of 20% and disease control rate of 70%. Median progression-free survival was 12.4 months (95% CI 6.4 to 24.9). The ORR for Programmed Death-Ligand 1 (PD-L1)-positive patients was 70% compared with 46% for PD-L1-negative patients. The combination was well tolerated, and side effects were similar to what was expected from single agent (P).ConclusionIn this study, the combination of indoximod and (P) was well tolerated and showed antitumor efficacy that is worth further evaluation in selected patients with advanced melanoma.

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.

Objectives: To compare the efficacy and safety of algenpantucel-L [HyperAcute-Pancreas algenpantucel-L (HAPa); IND# 12311] immunotherapy combined with standard of care (SOC) chemotherapy and chemoradiation to SOC chemotherapy and chemoradiation therapy alone in patients with borderline resectable or locally advanced pancreatic ductal adenocarcinoma (PDAC). Summary Background Data: To date, immunotherapy has not been shown to benefit patients with borderline resectable or locally advanced unresectable PDAC. HAPa is a cancer vaccine consisting of allogeneic pancreatic cancer cells engineered to express the murine α(1,3)GT gene. Methods: A multicenter, phase 3, open label, randomized (1:1) trial of patients with borderline resectable or locally advanced unresectable PDAC. Patients received neoadjuvant SOC chemotherapy (FOLFIRINOX or gemcitabine/nab-paclitaxel) followed by chemoradiation (standard group) or the same standard neoadjuvant regimen combined with HAPa immunotherapy (experimental group). The primary outcome was overall survival. Results: Between May 2013 and December 2015, 303 patients were randomized from 32 sites. Median (interquartile range) overall survival was 14.9 (12.2–17.8) months in the standard group (N = 158) and 14.3 (12.6–16.3) months in the experimental group (N = 145) [hazard ratio (HR) 1.02, 95% confidence intervals 0.66–1.58; P = 0.98]. Median progression-free survival was 13.4 months in the standard group and 12.4 months in the experimental group (HR 1.33, 95% confidence intervals 0.72–1.78; P = 0.59). Grade 3 or higher adverse events occurred in 105 of 140 patients (75%) in the standard group and in 115 of 142 patients (81%) in the experimental group (P > 0.05). Conclusions: Algenpantucel-L immunotherapy did not improve survival in patients with borderline resectable or locally advanced unresectable PDAC receiving SOC neoadjuvant chemotherapy and chemoradiation. Trial Registration: ClinicalTrials.gov Identifier: NCT01836432

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.

LESSONS LEARNEDHyperAcute Renal immunotherapy was well tolerated and demonstrated antitumor activity in patients requiring salvage-line treatment for metastatic renal cell carcinoma (mRCC).HyperAcute Renal immunotherapy was safely administered with concomitant salvage-line treatments for mRCC, and it may be a candidate for inclusion in novel combinations for salvage treatment of mRCC because of its unique mechanism of action.BACKGROUNDHyperAcute Renal (HAR) immunotherapy exploits a naturally occurring barrier to xenotransplantation and zoonotic infections in humans to immunize patients against metastatic renal cell carcinoma (mRCC) cells. HAR consists of two allogeneic renal cancer cell lines genetically modified to express α(1,3)Gal, to which humans have an inherent pre-existing immunity.METHODSPatients with refractory mRCC were eligible for this phase I dose-escalation trial. Concomitant treatment was permitted after the initial 2 months of HAR monotherapy. HAR was injected intradermally weekly for 4 weeks then biweekly for 20 weeks, totaling 14 immunizations. The primary endpoint was safety and determination of a maximum tolerated dose (MTD).RESULTSAmong 18 patients enrolled, two grade 3 adverse events (AEs) were attributed to HAR, lymphopenia and injection site reaction, and no grade 4/5 AEs occurred. The recommended phase II dose (RP2D) was 300 million cells. One patient had a partial response and eight patients had stable disease, for a disease control rate of 50% (9/18). Median overall survival with low-dose HAR was 14.2 months and was 25.3 months with high-dose HAR.CONCLUSIONIn pretreated mRCC, HAR immunotherapy was well tolerated and demonstrated antitumor activity. HAR immunotherapy may be a candidate for inclusion in novel combinations for salvage treatment of mRCC.

590 Background: HyperAcute Renal (HAR) immunotherapy consists of two allogeneic renal cancer cell lines that have been genetically modified to express α(1,3)Gal, to which humans have an inherent pre-existing immunity. A previous report demonstrated that HAR is well tolerated in pts with mRCC (2017 GUASCO, abstract: 528). Herein, we report the efficacy of HAR immunotherapy in mRCC. Methods: Pts with refractory clear-cell mRCC were eligible for this phase 1 dose-escalation trial. Concomitant treatment (Rx) with other approved agents was permitted after initial 2 months (m) of HAR monotherapy. The trial followed a standard 3+3 design with cells injected intradermally weekly for 4 weeks then biweekly injections for 10 immunizations (150 x106cells then escalated to 300 x106cells). Co-primary objectives were safety and efficacy. Results: A total of 18 patients were enrolled (4 low dose, 14 high dose) between 06/2015 to 07/2016. Patients received a median of 1 systemic Rx prior to HAR immunotherapy, with 8 patients receiving 2 or more prior agents. IMDC risk categories at the time of initial metastatic disease were: favorable risk (33%), intermediate risk (66%), poor risk (0%). The ORR was 0% with a disease control rate of 50%. Median PFS for patients treated with HAR immunotherapy was 2.0 months (m) (range 1.7-30.3 m). For patients receiving the low dose HAR, median overall survival (OS) was 14.2 m (range 3.6-21.6 m), while median OS for high dose HAR was 25.3 m (5.8-29.3 m). At the time of data cutoff in 09/2018, 7 patients were still living. Detailed clinical data will be presented in the meeting. Conclusions: HAR immunotherapy in refractory mRCC was well tolerated and demonstrated potential efficacy for OS similar to currently approved salvage-line Rx. With a unique mechanism of action, HAR immunotherapy may be a candidate for inclusion in novel combinatorial regimens being developed in salvage therapy setting in pts with mRCC. Clinical trial information: NCT02035358.

Background: NLG207 (formerly CRLX101) is an investigational nanoparticle-drug conjugate (NDC) composed of a cyclodextrin-based polymer backbone linked to camptothecin, a topoisomerase-1 inhibitor. NDCs enhance drug delivery to tumors where gradual payload release inside cancer cells augments antitumor activity while reducing toxicity. The RP2D of CRLX101 was previously set at 15 mg/m2 Q2W in combination with 80 mg/m2 weekly paclitaxel in Phase I and reported at ESMO 2016. This abstract will include data from both the Phase Ib and the phase II expansion. Methods: In this Phase II single arm study, patients were treated with NLG207 (Q2W) and 80 mg/m2 paclitaxel (3 wks on/1 wk off) repeated q 28 days until progression or toxicity. Primary objective was overall response (CR + PR) via RECIST v1.1 in women with recurrent platinum resistant epithelial ovarian, fallopian tube or primary peritoneal cancer. Progression free survival (PFS), duration of response (DOR), and safety were also assessed. Results: Thirty patients were enrolled and all completed at least one cycle. Median age was 62 (44-76) years. 57% of patients received ≥3 prior therapies. There were 8/30 confirmed responses for an overall response rate (ORR) of 26.7% (95% CI 14.2%, 44.4%), including one complete response (CR). ORR for platinum resistant patients (n=17) was 23.5% (4/17) vs. 30.8% (4/13) for those who were platinum sensitive (n=13) to their most recent platinum regimen. Best response (including unconfirmed) for platinum resistant patients was 41.2% (7/17) including 7 PRs. Five additional platinum resistant patients achieved stable disease (SD). Median PFS (mPFS) for all study patients was 5.4 months. The mPFS was similar for both platinum resistant at 5.5 months and platinum sensitive at 5.4 months. Median DOR was 7.2 months for platinum resistant patients vs. 4.7 months for the platinum sensitive group. The most common grade 3/4 adverse events (AEs) attributed to study treatment were: decreased neutrophil count (13 patients, 43%), anemia (3 patients, 10%), hematuria (2 patients, 7%); urinary tract infection (UTI), cystitis, hypertension, and hypokalemia (all seen in 1 patient, 3%). PK data will be included in the full presentation. Conclusions: NLG207 is a potentially best-in-class topoisomerase 1 inhibitor with demonstrated antitumor activity in recurrent ovarian cancer including those who have become resistant to platinum therapy. AE profile of this combination is consistent with that seen for paclitaxel as a single agent except for cystitis, hematuria and UTI. It was well-tolerated in combination with weekly paclitaxel in heavily pre-treated patients. NLG207 warrants further investigation in combination therapy regimens for recurrent ovarian, fallopian tube or primary peritoneal cancer, particularly in platinum resistant patients. Citation Format: Linda Duska, David M. O'Malley, Carolyn Krasner, Russell J. Schilder, Cara Mathews, Kathleen Moore, Premal Thaker, Austin Miller, Christopher Purdy, A.J. Leyco, Christopher Smith, Deborah Mercier, Lucinda Tennant, Eugene Kennedy, Nicholas Vahanian, Charles Link. A Phase II study of NLG207 (formerly CRLX101) in combination with weekly paclitaxel in patients with recurrent or persistent epithelial ovarian, fallopian tube or primary peritoneal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT151.

4015 Background: The indoleamine 2,3-dioxygenase (IDO) pathway is a key counter-regulatory mechanism that is exploited by tumors to prevent and evade anti-tumor immunity. Inhibitors of the IDO pathway, such as indoximod, are an increasingly validated class of potential cancer therapeutics. The combination of gemcitabine (G) and nab-paclitaxel (N) is a current SOC for metastatic pancreas cancer (MPC). Pre-clinical models have demonstrated synergy between indoximod and chemotherapy. Methods: Single arm study with indoximod (1200mg BID continuous) plus G / N (1000mg/m2 / 125mg/m2 q week x3 per 4-week cycle). Patients had treatment naïve MPC or 1st line therapy after previous resection and adjuvant therapy. Treatment continued until disease progression or toxicity. Primary endpoint was an improvement in median overall survival (mOS) from a historical 8.5 months for G / N to 12.1 months (hazard ratio (HR) of 0.70). Secondary endpoints included overall response rate (ORR) by site review RECIST 1.1. An expansion cohort enrolled patients undergoing pre and on-treatment (end Cycle 2) tumor biopsies. Results: A total of 135 patients initiated treatment in Phase 2 including 36 in the biopsy group. 104 were efficacy evaluable (EE) per the pre-specified definition of completing one cycle of therapy with one on-treatment imaging study. The ORR in the EE was 46.2% (48/104) with 1% CR (1/104) and 45.2% PR (47/104). The mOS in the EE was 10.9 months. Combination was generally well tolerated with fatigue, nausea, and anemia being the most commonly observed adverse events. Immunologic data by immunohistochemistry from biopsy samples (n = 11) indicate responders have increased intra-tumoral CD8 density after 2 cycles of therapy compared to non-responders (p = 0.030). Conclusions: EE patients had a mOS of 10.9 months and ORR of 46.2%; responding patients had an increased intra-tumoral CD8 density. The study did not meet the pre-specified primary goal of a 30% reduction in HR. However, the combination demonstrated activity with a promising ORR and immunologic correlation with response. These data support the continued development of combination indoximod immuno-chemotherapy for MPC. Clinical trial information: NCT02077881.

Indoximod is an IDO pathway inhibitor with a differentiated mechanism of action directly targeting immune cells to reverse the immune suppression generated by tumors. An ongoing phase-1b/2a study (NCT02502708) using indoximod in combination with temozolomide and/or re-irradiation for children with relapsed/refractory pediatric brain tumors has established the recommended phase-2 dose (RP2D) of indoximod for this regimen and has shown this approach to be well-tolerated and feasible in these highly complex and fragile patients. Diffuse Intrinsic Pontine Glioma (DIPG) is an FDA-designated orphan disease, with no curative treatment options and dismal prognosis. The primary hypothesis is that addition of indoximod-based immunotherapy to standard-of-care radiation, followed by immuno-chemotherapy with indoximod plus temozolomide will improve objective response rates, 12-month event-free survival, and median overall survival. Newly-diagnosed DIPG patients age 3 to 21 years are treated with indoximod (RP2D) in combination with conformal radiation therapy (54 Gy), followed by cyclic immune-chemotherapy using indoximod (RP2D=38.4 mg/kg/day divided BID throughout each cycle) combined with temozolomide (200 mg/m2/day, days 1-5 of each 28-day cycle). Up to 30 patients may be enrolled. The trial is ongoing. At this time, we have enrolled the first 6 newly-diagnosed DIPG patients. At the end of the indoximod plus radiation block, the first two patients had objective tumor response without any significant adverse events to date. Interim outcome and safety data for the first 8 months of accrual will be presented.

Abstract Background Successful allogeneic stem cell transplantation (HSCT) in treatment of patients (pts) with acute myeloid leukemia (AML) is dependent upon graft-versus-leukemia, suggesting that intact immune surveillance is essential for eradicating minimal residual disease. Myeloblast-induced T-cell tolerance through overexpression of indoleamine 2,3-dioxygenase (IDO) is thought to play a significant role in immune evasion through upregulation of tryptophan (Trp) catabolism and kynurenine production, resulting in a Trp-poor environment that leads to immune system suppression. Indoximod is a small-molecule inhibitor of the IDO pathway that acts directly on immune cells to reverse IDO pathway-mediated suppression. We are assessing the safety and preliminary efficacy of indoximod in combination with standard induction chemotherapy in patients (pts) with newly diagnosed AML. Methods In this open-label, multicenter, phase 1 study (NCT02835729), eligible pts with newly diagnosed AML were treated with indoximod in combination with induction chemotherapy (idarubicin 12 mg/m2/d x3 days with cytarabine 100 mg/m2/d x7 days). Using a "3+3" design, indoximod (600 mg [dose level 0], 1000 mg [dose level 1], 1200 mg [dose level 2]) was given orally every 8 hours (Q8h) starting on day 9 of induction. Regimen limiting toxicity (RLT) was defined as any ≥ grade 3 non-hematologic adverse event (AE) that was not incontrovertibly related to the underlying AML or cytarabine or idarubicin. After induction, pts received up to 4 cycles of high dose cytarabine (HiDAC) consolidation while continuing indoximod. Patients continued on maintenance indoximod for up to 6 months from completion of consolidation therapy. Indoximod was discontinued 4 weeks prior to HSCT in eligible patients and not restarted as maintenance post-HSCT. Results As of July 15, 2018, 31 pts were enrolled (median age 55 years, range 18-78; 77% male). Six patients did not proceed with study therapy due to a diagnosis of acute promyelocytic leukemia, issues with medical insurance coverage, consent withdrawal, critical illness, and intestinal myeloid sarcoma preventing oral intake. Pts who received ≥1 dose of indoximod were included in the intention-to-treat (ITT) analysis (n=25), and pts who received ≥80% of their scheduled indoximod doses were included in the per-protocol (PP) analysis (n=19). Reasons for not completing ≥80% of indoximod doses were: consent withdrawal (n=3), inability to swallow (n=2) and physician decision (n=1). Of the 19 PP patients, 16 (84%) had either unfavorable karyotype or adverse mutation profile and 3 (16%) had secondary AML (s-AML). Indoximod combined with induction chemotherapy was well tolerated; no RLT was observed. The most frequent grade ≥3 non-hematologic treatment-emergent AEs in the ITT population, regardless of attribution, were febrile neutropenia (60%), hypoxia (16%), atrial fibrillation (12%), pneumonia (12%), hypocalcemia (12%), and hypotension (12%). Among 25 ITT pts, 21 (84%) achieved a remission (CR/CRh/CRi/CRp), and 15 of 19 (79%) in the PP analysis achieved remission. Among 12 pts with measurable residual disease (MRD) available in remission, 10 (83%) had MRD <0.02% (MRD-neg). Eleven of 19 pts (58%) received ≥1 cycle of HiDAC and 5 (26%) received maintenance indoximod. All 11 patients who received HiDAC #1 became MRD-neg. Median relapse-free and overall survival have not been reached. IDO Composite Scores in bone marrow were calculated by multiplying percentage of stained mononuclear cells by grade of staining intensity determined by 3 independent pathologists. Median composite IDO1 score in tested pt samples (n=11) was 0.76 (range, 0.1-2.2). Expression of IDO1 mRNA at baseline varied significantly among patient samples analyzed (fold changes (FC) range: 0.1-84, normalized to β-Actin expression). IDO1 mRNA was significantly upregulated in post-induction samples compared to baseline (FC range: 1.7-248) in 10 out of 12 paired samples. Conclusions Indoximod is well tolerated in combination with standard AML induction therapy. Rates of morphologic response and of MRD-neg status are very promising. The recommended phase 2 dose (RP2D) was 1200 mg oral Q8h and a placebo-controlled randomized phase 2 study is under development. Disclosures Emadi: NewLink Genetics: Research Funding. Loken:Hematologics, Inc: Employment, Equity Ownership. Kennedy:NewLink Genetics: Employment, Equity Ownership. Link:NewLink Genetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Munn:NewLink Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The IDO pathway mediates immunosuppressive effects by metabolizing tryptophan (Trp) into kynurenine (Kyn). The depletion of Trp stimulates downstream signaling through nutrient sensors GCN2 and mTOR, while the production of Kyn stimulates signaling through the aryl-hydrocarbon receptor (AHR) transcription factor. The activation of these signaling pathways has pleiotropic effects on immune cells, including influencing the differentiation of dendritic cells (DCs), helper T cells, and regulatory T cells (Treg) as well as enhancing the proliferation of effector T cells and Treg. Indoximod has been demonstrated to relieve IDO-mediated immunosuppression in vitro and in vivo, by creating an artificial Trp-sufficiency signal that bypasses activation of GCN2 and inhibition of mTOR in conditions of Trp deprivation. We hypothesized that indoximod9s activity could also include the disruption of AHR activation by Kyn and other Trp catabolites, causing differential AHR signaling and transcriptional activity, resulting in an augmented antitumor immune response. We observed that indoximod activates AHR-dependent transcriptional activity in HepG2 cells as evidenced by increased AHR-driven luciferase and endogenous CYP1A1 activity. AHR activation by indoximod was also observed in primary human T cells, as measured by the induction of CYP1A1 mRNA. In a Kyn-driven Treg differentiation assay, indoximod altered transcription of genes associated with T helper and Treg phenotypes. Indoximod induced upregulation of Rorc expression, a TH17-associated transcription factor, while concurrently downregulating transcription of Foxp3, the master transcription factor of Treg cells. These effects were reverted by an AHR inhibitor. Consistent with the gene expression profiles, indoximod shifted the cellular phenotype from Foxp3+ Treg toward Th17-producing CD4 helper T cells. Transcription of Ido1 is controlled by IFNγ and AHR-response elements in its promoter. Consistent with the role of indoximod in blocking the IDO pathway, we observed that indoximod downregulated the expression and function of IDO in in vitro derived plasmacytoid DC that normally would express IDO under the same differentiation conditions. When these in vitro differentiated human pDCs were used in an MLR culture, the indoximod-induced downregulation of IDO resulted in decreased Kyn production and increased T cell proliferation. Moreover, indoximod treatment of tumor-bearing mice decreased expression of IDO in the pDC in tumor-draining LN. Together, these data suggest that indoximod modulates AHR signaling to exert multiple immunomodulatory effects, including a shift from suppressive Foxp3+ Treg toward TH17 helper T cells as well as the downregulation of IDO expression in pDC, contributing to enhanced antitumor immunity. Citation Format: Erik L. Brincks, James Adams, Michael Essmann, Benjamin A. Turner, Lifu Wang, Jiyuan Ke, Agnieszka Marcinowicz, Nicholas Vahanian, Charles J. Link, Mario R. Mautino. Indoximod modulates AhR-driven transcription of genes that control immune function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3753.

Abstract BACKGROUND: In theory, combining immunotherapy with radiation (RT) and chemotherapy is an appealing therapeutic strategy that may offer potential synergistic benefits. The indoleamine 2,3-dioxygenase (IDO) pathway regulates inflammation and immune responses to apoptotic cells. Indoximod reverses the profound immunosuppression created by IDO in the tumor microenvironment. METHODS: 29 pediatric patients with recurrent or progressive malignant brain tumors were treated in two separate indoximod dose-escalation cohorts. Patients eligible for re-irradiation (re-RT) (17/29) were treated with conformal RT (15-54 Gy) in combination with indoximod, followed by indoximod with cyclic temozolomide (200 mg/m2/day for the first 5 days of each 28-day cycle). Patients not eligible for RT received indoximod plus cyclic temozolomide. After initial response or disease stabilization (at least 4 months), patients who showed progression were offered planned re-RT to escape lesions, surgical debulking where indicated, and cross-over to an alternative chemotherapy regimen (oral metronomic cyclophosphamide plus etoposide) while continuing indoximod immunotherapy. Time to Regimen Failure (TTRF) was used to capture time on study. RESULTS: In aggregate, 29 relapsed patients had a median PFS of 6.2 months, median time on study (TTRF) was 11.7 months, and 9/29 are continuing treatment. Patients given RT had median TTRF 13.5 months vs. 4.3 months for no RT. Patients not eligible for conventional full-dose re-RT (>50 Gy) received lower-dose RT (=<30 Gy) not attempting to target all disease where that would be toxic. 9 of 17 patients received lower-dose, restricted-field RT and median TTRF was 16 months versus 13 months for full-dose RT. Based on these results, a cohort of newly-diagnosed patients with diffuse intrinsic pontine glioma (DIPG) was opened using indoximod during front-line RT (54 Gy) followed by maintenance indoximod plus temozolomide. To date 6 newly-diagnosed DIPG patients have enrolled, all with bulbar signs and bulky disease on MRI at study entry. The first 3 newly-diagnosed DIPG patients have completed induction radio-immunotherapy which was well tolerated with symptomatic improvement in all patients, radiographic responses in 3/3, and near resolution of one tumor at the end of radiotherapy. An additional patient with progressive DIPG received re-RT (27 Gy) combined with indoximod, which was tolerated well with symptomatic improvement and objective tumor reduction on post-RT MRI. CONCLUSIONS: Indoximod was well-tolerated in combination with RT and chemotherapy. TTRF compares favorably with historical controls. Lower-dose, restricted-field RT may be beneficial when combined with indoximod in relapse cases. Front-line studies have begun in a cohort of DIPG patients. Citation Format: Theodore S. Johnson, Dolly Aguilera, Ahmad Al-Basheer, Robert C. Castellino, Bree R. Eaton, Natia Esiashvili, Nicholas Foreman, Ian M. Heger, Eugene P. Kennedy, Charles J. Link, William Martin, Eric K. Ring, Ramses F. Sadek, Amy Smith, Vahanian N. Vahanian, Tobey J. MacDonald, David H. Munn. Front-line therapy of DIPG using the IDO pathway inhibitor indoximod in combination with radiation and chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT004.

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.

Background: The indoleamine 2,3-dioxygenase (IDO) pathway is a key counter-regulatory mechanism that normally inhibits immune responses when appropriate. In the setting of cancer, IDO pathway-mediated immune suppression is exploited by tumors in order to prevent and defeat anti-tumor immunity. Small-molecule inhibitors of the IDO pathway, such as indoximod, are an increasingly validated class of potential cancer therapeutics. Additionally, pre-clinical tumor models have shown complementary effects with indoximod / anti-PD1 checkpoint inhibitor treatment combinations. A clinical trial was developed based upon these data. Methods: Upon successful completion of a Phase 1b dose escalation cohort, metastatic melanoma patients were enrolled in a single arm Phase 2 trial evaluating the addition of indoximod to standard of care checkpoint inhibitors approved for melanoma. Treating physicians were allowed to administer their choice of approved checkpoint inhibitor. The large majority of patients received indoximod with pembrolizumab and this interim report is limited to those patients. Indoximod was administered continuously in 21 days cycles (1200mg po twice daily) concurrently with pembrolizumab (3mg/kg q21 days). Study endpoint is best overall response (objective response rate (ORR) = complete response rate (CR) + partial response rate (PR)) per site reported RECIST criteria. Results: At time of data cut-off, 60 patients had received indoximod /pembrolizumab and were evaluable for response, defined as having at least one follow-up imaging study performed. The ORR was 52% (31/60) with a CRR of 8% (5/60). The combination was well tolerated. The most frequently reported adverse events (regardless of attribution), occurring in ≥ 20% of subjects, were fatigue, diarrhea, nausea, arthralgia, headache, cough, rash, pruritus, and hypertension. The most frequently reported laboratory abnormalities (regardless of attribution), were anemia (17%) and hyperglycemia (17%). Conclusions: The interim analysis of the combination of indoximod and pembrolizumab demonstrates an ORR of 52% which compares favorably with the established ORR for pembrolizumab alone. Updated data to be presented. NCT02073123. Citation Format: Yousef Zakharia, Robert McWilliams, Monaster Shaheen, Kenneth Grossman, Joseph Drabick, Mohammed Milhem, Olivier Rixie, Samir Khleif, Ryan Lott, Eugene Kennedy, David Munn, Nicholas Vahanian, Charles Link. Interim analysis of the Phase 2 clinical trial of the IDO pathway inhibitor indoximod in combination with pembrolizumab for patients with advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT117. doi:10.1158/1538-7445.AM2017-CT117

Abstract IDO1 catalyzes degradation of tryptophan (Trp) into kynurenines, which plays an important role in the regulation of immune responses by triggering anergy on reactive effector T cells and by modulating differentiation and activation of regulatory T cells (Treg). Indoximod has been demonstrated to relieve IDO-mediated immunosuppression in vitro and in vivo, by creation of an artificial Trp-sufficiency signal that bypasses activation of GCN2 and inhibition of mTOR in conditions of Trp deprivation. Inhibition of the IDO pathway by indoximod in combination with immune-stimulatory treatments leads to increased T cell proliferation, Treg reprogramming and antitumor effect. Indoximod has demonstrated an excellent safety profile in human clinical trials and is being dosed orally at 1200 mg bid. Increases in doses above this level do not generally result in increased plasma concentration or drug exposure due to limiting dose-dependent oral bioavailability. Animal models suggest that increased therapeutic benefit could be achieved at higher levels of exposure. For this reason, we synthesized and tested the pharmacokinetic profile of several indoximod prodrugs in mice, rat and monkeys in both liquid and capsule formulations. We selected clinical development drug candidate NLG802, which increases indoximod exposure and plasma concentration ~ 2-fold in rats and ~ 5-fold in monkeys dosed with capsules of comparable formulation to that being used in clinical trials. NLG802 is rapidly absorbed and metabolized in vivo to indoximod. NLG802 DMPK profile and GLP toxicology studies have been carried out in rats and monkeys suggesting a safe toxicological profile at predicted therapeutic doses. In a preclinical tumor model of mice bearing established B16F10 tumors, administration of NLG802 markedly enhanced the anti-tumor responses of naïve, resting pmel-1 cells to vaccination with cognate hgp100 peptide plus CpG-1826 in IFA. In this tumor model, NLG802 plus pmel-1/vaccine produced significant tumor size reduction within 4 days of vaccination. Moreover, the tumor response was achieved at lower doses than equivalent molar doses of indoximod. In conclusion, NLG802 is a prodrug of indoximod predicted to increase clinical drug exposure to indoximod above the current achievable levels and will soon enter Phase 1 safety testing in oncology clinical trials. Citation Format: Mario R. Mautino, Sanjeev Kumar, Hong Zhuang, Jesse Waldo, Firoz Jaipuri, Hima Potturi, Erik Brincks, James Adams, Agnieszka Marcinowicz, Clarissa Van Allen, Nicholas Vahanian, Charles J. Link. A novel prodrug of indoximod with enhanced pharmacokinetic properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4076. doi:10.1158/1538-7445.AM2017-4076

528 Background: HyperAcute Renal (HAR) immunotherapy consists of two allogeneic renal cancer cell lines that have been genetically modified to express the carbohydrate α(1,3)Gal, to which humans have an inherent pre-existing immunity. HAR is designed to leverage this mechanism to educate the immune system towards antigens expressed by the patient’s own tumor cells. Methods: This study is a Phase 1 dose escalation trial of HAR for patients with Renal cell carcinoma (RCC). Eligible patients had recurrent or metastatic RCC with a clear cell component. Trial was a standard 3+3 design with patients receiving weekly intradermal injection of HAR (150 x106 cells/300 x106 cells intradermally) for 4 weeks followed by biweekly injections for 10 weeks. After the initial determination of dose-limiting toxicity, an expansion cohort up to 14 patients was enrolled. The primary endpoint was safety and determination of a maximum tolerated dose (MTD). Secondary endpoints are efficacy and immunological correlates. Results: A total of 18 patients (4 low dose, 14 high dose) were enrolled. The MTD was set at 300 x106 cells intradermally with no dose limiting toxicity (DLT). The most frequently reported adverse events (regardless of attribution), occurring in ≥ 20% of subjects, were abdominal pain, fatigue, pain, and injection site reactions. The most frequently reported laboratory abnormality (regardless of attribution) was anemia in 20% of subjects. There have been 5 reported serious adverse events, none deemed related to HAR. Conclusions: RCC is considered an immunogenic tumor based on its response rate to immune checkpoint blockade and IL-2, occasional spontaneous regression, and the high level of tumor T cell infiltration. HAR was well tolerated in this patient population. Therefore, RCC is an appropriate tumor type to target with combination immunotherapy including HAR. Updated results will be presented. Clinical trial information: NCT02035358.

Highly pathogenic avian influenza represents a severe public health threat. Over the last decade, the demand for highly efficacious vaccines against avian influenza viruses has grown, especially after the 2013 H7N9 outbreak in China that resulted in over 600 human cases with over 200 deaths. Currently, there are several H5N1 and H7N9 influenza vaccines in clinical trials, all of which employ traditional oil-in-water adjuvants due to the poor immunogenicity of avian influenza virus antigens. In this study, we developed potent recombinant avian influenza vaccine candidates using HyperAcute™ Technology, which takes advantage of naturally-acquired anti-αGal immunity in humans. We successfully generated αGal-positive recombinant protein and virus-like particle vaccine candidates of H5N1 and H7N9 influenza strains using either biological or our novel CarboLink chemical αGal modification techniques. Strikingly, two doses of 100 ng αGal-modified vaccine, with no traditional adjuvant, was able to induce a much stronger humoral response in αGT BALB/c knockout mice (the only experimental system readily available for testing αGal in vivo) than unmodified vaccines even at 10-fold higher dose (1000 ng/dose). Our data strongly suggest that αGal modification significantly enhances the humoral immunogenicity of the recombinant influenza vaccine candidates. Use of αGal HyperAcute™ technology allows significant dose-sparing while retaining desired immunogenicity. Our success in the development of highly potent H5N1 and H7N9 vaccine candidates demonstrated the potential of αGal HyperAcute™ technology for the development of vaccines against other infectious diseases.