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Tumor-associated macrophages (TAMs) play a role in cancer progression and are associated with Sorafenib resistance in hepatocellular carcinoma (HCC)1. D-4559 is a new potent macrophage switching nanomedicine technology that selectively inhibits VEGF receptor tyrosine kinases (VEGFR1, 2, 3) in TAMs leading to a functional reprogramming of TAMs toward a pro-inflammatory activated phenotype. Here, we evaluate the effect of D-4559 on the M1 and M2 polarization of TAMs and its anti-tumor efficacy in a murine HCC tumor model. In vivo efficacy of D-4559 was examined in the subcutaneous Hepa 1-6 liver tumor model in C57BL/6 mice. Animals (n=15/group) were treated with D-4559 (i.p., 200 mg/kg daily) and free drug Sorafenib (p.o., 40 mg/kg daily) as a positive control for 4 weeks. The treatment started when the mean tumor size reached approximately 100 mm3, then the animals were randomly allocated into study groups. The day of randomization and treatment was denoted as day 0. Tumor sizes were measured using a caliper for 27 days, M1/M2 macrophage polarization was examined by flow cytometry at day 16, and cytokine biomarkers were evaluated with MSD Cytokine Multiplex Assay at day 16 and 27.D-4559 significantly reduced Hepa 1-6 tumor growth (**p References: 1. Wang et al, Journal of Biomedical Science (2022) 2. Hayashi, T., et al. BMC Cancer 17, 870 (2017) 3. Li, H., et al., Translational Cancer Research 8, 2 (2019) Citation Format: Naze G. Avci, Tony Wu, Susan E. Alters, Emily Zhang, Jinping Liu, Dong Huang, Natacha Le Moan, Jeffrey L. Cleland. A potent macrophage switching drug D-4559 reduces tumor GROWTH in a hepatocellular carcinoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB208.

Advanced or metastatic breast cancers represents a large patient population. Targeted therapies following hormonal or chemotherapeutic approaches offer some benefit. Subsequently, molecular profiling has identified over 100 mutations that render inhibitor resistance, creating challenges for physicians, patients, and the healthcare system broadly. While advances in therapeutic options have improved treatment outcomes by prolonging PFS with better QOL, long-term solutions remain a challenge for the majority of patients. The aim of our work is to drive strong mechanistic responses in this large patient population by developing targeted therapies to a key fundamental mechanism at the convergence of many oncogenic signaling pathways through which resistance arises. Our work in established cell lines and primary patient derived organoids demonstrated potent and complete responses in breast cancers that represent patient populations where unmet needs remain high including ER positive, TNBC, PTEN and RB null molecular profiles. The regulation of CAP dependent protein translation is a central element in oncogenesis. One of a family of CAP binding proteins, eIF4E serves to anchor the m7GTP CAP of RNA into several protein complexes and regulates multiple aspects of mRNA processing such as nuclear export and ribosomal translation. Previous communications revealed our eIF4E regulators potently cause cell death in primary breast cancer organoid models differentially to models derived from healthy tissue. We have expanded our efforts following our observations of both rapid apoptosis and differential sensitivity of healthy cells. In vitro, in breast cancer cells we observed stable responses in mechanistic markers of pharmacodynamic response, including phospho-Rb, with short term drug incubation. In vivo, in a ZR-75-1 breast CDX model, intermittent dosing (QW) of our lead compound resulted in strong tumor growth inhibition (>95%). Consistent with growth impacts, we observed stable pharmacodynamic marker (p-Rb) changes in tumor tissue at 24 and 48 hours. To understand the impact on immune cells, we evaluated our regulators against primary cells including PBMCs, CD4+ T cells, and bone marrow mononuclear cells. Our eIF4E regulators did not impair survival of these cells up to a maximum tested dose of 25 micromolar. Primary CD4+ T cells were also functional in the presence of eIF4E regulators up to 5 micromolar, which included assessments of CD3/CD28 stimulated cytokine production and proliferative responses. Our findings suggest that allosteric modulation of eIF4E provides a unique and efficient way to address multiple resistance mechanisms while sparing normal immune cellular function. Thus, our allosteric eIF4E regulators represent a potential beneficial therapeutic approach to address multiple resistant cancer patient populations and fulfill the promise of this elusive target. Citation Format: Lisa-Marie Sturla, Xuemei Zhang, Katherine Bowdish, Susan E. Alters, Christopher VanDeusen. eIF4E allosteric regulators cause rapid commitment to apoptosis in cancer cells while sparing immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1624.

Metabolic remodeling contributes to the development and progression of some cancers and exposes them to vulnerabilities, including specific nutrient dependencies that can be targeted therapeutically. Arginine is a semiessential amino acid, and several cancers are unable to endogenously synthesize sufficient levels of arginine for survival and proliferation, most commonly due to reduced expression of argininosuccinate synthase (ASS1). Such cancers are dependent on arginine and they can be targeted via enzyme-mediated depletion of systemic arginine. We report the preclinical safety, antitumor efficacy, and immune-potentiating effects of pegzilarginase, a highly potent human arginine-degrading enzyme. Toxicology studies showed that pegzilarginase-mediated arginine depletion is well tolerated at therapeutic levels that elicit an antitumor growth effect. To determine which tumor types are best suited for clinical development, we profiled clinical tumor samples for ASS1 expression, which correlated with pegzilarginase sensitivity in vivo in patient-derived xenograft (PDx) models. Among the histologies tested, malignant melanoma, small cell lung cancer and Merkel cell carcinoma had the highest prevalence of low ASS1 expression, the highest proportion of PDx models responding to pegzilarginase, and the strongest correlation between low or no ASS1 expression and sensitivity to pegzilarginase. In an immune-competent syngeneic mouse model, pegzilarginase slowed tumor growth and promoted the recruitment of CD8+ tumor infiltrating lymphocytes. This is consistent with the known autophagy-inducing effects of arginine depletion, and the link between autophagy and major histocompatibility complex antigen presentation to T cells. Our work supports the ongoing clinical investigations of pegzilarginase in solid tumors and clinical combination of pegzilarginase with immune checkpoint inhibitors.

Previous studies have demonstrated hydroxyl dendrimers (HDs) are selectively taken up by tumor associated macrophages (TAMs) after a single administration. HDs have also been observed to cross the blood brain barrier in six animal species including dogs and monkeys. The treatment of brain tumors and metastases is hampered by the inability to deliver radiotherapy or cancer agents selectively to the tumor. HDs of two different generations were prepared (HD4 and HD6; both smaller than antibodies) and DOTA was conjugated to the HD through linker arms attached to the hydroxyls on the dendrimer surface. 111In radiolabeling of HD4- and HD6-DOTA resulted in >95% radiochemical purity in both cases and specific activities of 0.621 mCi/mg and 0.638 mCi/mg, respectively. GL-261-luc cells, a syngeneic glioblastoma, were implanted intracranially in 8 female albino C57.bl/6 mice. Tumors were 15-60 mm3 at time of dosing. Mice received a single administration of HD4-DOTA-111In or HD6-DOTA-111In at a dose of approximately 0.5 mg/mouse and 0.3 mCi/mouse. Whole body SPECT/CT imaging was done over time up to 6 days post-dose in 3 animals per compound. The biodistribution and uptake in the brain tumor and other tissues was quantitatively measured using VivoQuant 4.0 (Invicro, Boston). Both HDs rapidly distributed in the systemic circulation after administration. Clearance appeared to be primarily via the kidneys based upon the fraction of injected dose observed in the bladder over time. The % injected dose (%ID)/g in the brain tumor was 2.67 ± 0.45 and 7.05 ± 0.53 at 24 h post-dose for the HD4 and HD6 compounds, respectively. The %ID/g in the contralateral brain section was 0.37 ± 0.17 and 0.90 ± 0.20 at 24 h post-dose for the HD4 and HD6 compounds, respectively. Tumor associated radioactivity persisted for the duration of the study (last time points: HD4, 96 h, 5.27 ± 2.04 %ID/g; HD6, 144 h, 3.20 ± 1.02 %ID/g). These results confirm previous studies in this model demonstrating selective tumor uptake and persistence with 58 fold greater radioactivity at the last time point in brain tumor compared to contralateral brain. The selectivity and prolonged duration of HDs in TAMs will enable the use of the same HD-DOTA constructs to deliver radiotherapeutic 90Y to brain tumors. The HD radiotherapy approach may eliminate the off-target toxicity of current ligand-based approaches. Citation Format: Rishi Sharma, Minghao Sun, Santiago Appiani, Richard Coelho, Patrick McConville, Susan Alters, Jeffrey L. Cleland. Selective targeting and imaging of orthotopic glioblastoma after a single systemic dose of a novel hydoxyl dendrimer radionuclide [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-097.

Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. Under conditions of elevated ROS, endogenous L-cysteine (L-Cys) production is insufficient for GSH synthesis. This necessitates uptake of L-Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(-) transporter. We show that administration of an engineered and pharmacologically optimized human cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet this treatment results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in both prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53-/- mice, which develop disease resembling human chronic lymphocytic leukemia. It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies.

A novel recombinant human growth hormone (rhGH) fusion protein (VRS-317) was designed to minimize receptor-mediated clearance through a reduction in receptor bind- ing without mutations to rhGH by genetically fusing with XTEN amino acid sequences to the N-terminus and the C-terminus of the native hGH sequence. Although in vitro potency of VRS- 317 was reduced approximately 12-fold compared with rhGH, in vivo potency was increased because of the greatly prolonged exposure to the target tissues and organs. VRS-317 was three- fold more potent than daily rhGH in hypophysectomized rats and fivefold more potent than daily rhGH in juvenile monkeys. In juvenile monkeys, a monthly dose of 1.4mg/kg VRS-317 (equivalent to 0.26mg/kg rhGH) caused a sustained pharmacodynamic response for 1 month equivalent to 0.05mg/kg/day rhGH (1.4mg/kg rhGH total over 28 days). In monkeys, VRS-317, having a terminal elimination half-life of approximately 110h, was rapidly and near-completely absorbed, and was well tolerated with no observed adverse effects after every alternate week subcutaneous dosing for 14 weeks. VRS-317 also did not cause lipoatrophy in pig and monkey studies. VRS-317 is currently being studied in GH-deficient patients to confirm the observa- tions in these animal studies. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:2744-2754, 2012

Background: Tumors with impaired arginine synthesis are susceptible to extracellular arginine depletion. We report results from the first-in-human phase I trial of pegzilarginase (AEB1102, Co-ArgI-PEG), a pegylated, recombinant, cobalt-substituted human arginase I (NCT02561234). Experimental design: Patients with metastatic solid tumors refractory to standard therapies were enrolled in 3+3 cohorts at increasing dose levels of weekly intravenous pegzilarginase. Primary endpoint was to determine the maximum tolerated dose (MTD). Additional endpoints included safety, pharmacokinetics, pharmacodynamics, immunogenicity, expression of arginine synthesis pathway enzymes in tumor tissue, and preliminary efficacy. Results: Forty patients were enrolled in 9 cohorts at doses from 0.01 to 0.40 mg/kg. The 3 most prevalent histologies (4 patients each) were colorectal and prostate adenocarcinoma, and uveal melanoma. Two dose limiting toxicities (DLT) were observed: G3 failure to thrive (0.33 mg/kg) and G3 maculopapular rash (0.40 mg/kg). After assessment of overall safety profile, the MTD was determined to be 0.33 mg/kg once weekly. Other related serious or ≥ grade 3 adverse events (AEs) not considered DLTs per protocol were hypophosphatemia (2 patients), and anemia, neutropenia, tremor, weakness, and transient hypertension (one occurrence each in a single patient). Pegzilarginase-related AEs in ≥10% of patients included nausea (9 patients with G1-2), stomatitis/mouth sores (8), fatigue (8), vomiting (6), pruritic, macular, or maculopapular rash (4, 1 G3), decreased appetite (4), and diarrhea (4). Pegzilarginase exposure was dose proportional on day 1 and day 29 for all doses starting at 0.02 mg/kg, and the mean half-life was constant at doses ≥ 0.04 mg/kg (33-55 hr after dose 5). On average, doses ≥0.18 mg/kg depleted arginine to ≤10% of baseline for up to 72 hrs. Analysis of the initial 27 patients revealed no anti-drug antibodies (ADAs). Median number of doses was 6.5 (range 1-20); 4 patients had stable disease after 8 weeks of treatment and continued in the trial for a total of 16-20 weeks. ASS1, ASL, and OTC expression levels varied across the different histologies. Conclusions: The MTD of pegzilarginase was 0.33 mg/kg, and the PK/PD profile supports once-weekly dosing with potential adjustable margin. The manageable safety profile, absence of ADA, and observation of stable disease supports further exploration as monotherapy and combination therapy. Expansion cohorts were initiated at MTD in specific histologies with high probability of low ASS1 expression (uveal melanoma, cutaneous melanoma and small-cell lung cancer [SCLC]). Based on pre-clinical data showing enhanced effects of PD-1 inhibition, a parallel study is investigating pegzilarginase in combination with pembrolizumab for patients with previously-treated SCLC. Citation Format: Drew W. Rasco, S. Gail Eckhardt, Diwakar Davar, Karl Lewis, Humberto Lara-Guerra, Susan E. Alters, Stephen Eckert, Scott W. Rowlinson, James E. Wooldridge, Richard D. Carvajal. Phase I dose escalation trial of pegzilarginase in patients with advanced solid tumors [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 CT030.

7031Background: In vitro studies demonstrate that AML cells are arginine auxotrophs and are metabolically vulnerable to arginine depletion (PMID: 24018014 & 25896651). To test the clinical utility ...

Purpose: The antitumor activities and pharmacokinetics of the hypoxia-activated cytotoxin AQ4N and its metabolites were assessed in several preclinical models of pancreatic cancers. Experimental Design: The cytotoxic effects of AQ4N prodrug and its bioreduced form, AQ4, were tested against multiple human tumor cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Nude mice bearing s.c. or orthotopically implanted human BxPC-3 or Panc-1 tumor cells were treated with AQ4N. Tumor growth inhibition, time to progression/end point, and liver metastasis were evaluated in treatment versus control groups. Plasma and tumor levels of AQ4N and its metabolites were quantitated by liquid chromatography-tandem mass spectrometry. Results: In contrast to AQ4N, the bioreduced AQ4 metabolite displayed potent cytotoxicity in many human tumor lines, including those derived from human pancreatic adenocarcinomas. Single-agent administration of AQ4N significantly delayed tumor growth, progression, and survival in a manner comparable with gemcitabine in multiple pancreatic tumor models in vivo. Survival increases were accompanied by a reduction in incidence and spread of liver metastasis. Quantitation of AQ4N and its metabolites in tumor-bearing mice showed that the prodrug is rapidly cleared from the circulation by 24 h and neither of the bioreduced metabolites was detected in plasma. In contrast, AQ4N readily penetrated BxPC-3 tumors and the cytotoxic AQ4 metabolite rapidly accumulated in tumor tissues at high levels in a dose-dependent fashion. Conclusion: AQ4N undergoes rapid and selective conversion into the potent antineoplastic metabolite AQ4 in tumors in vivo and provides proof of principle for the use of hypoxia-activated prodrugs in the treatment against pancreatic cancers.

In order to improve the in vitro and in vivo efficacy of an integrin antagonist (IA) of the extracellular domain of the alphavbeta3 integrin, a receptor upregulated on tumor neovasculature, the IA was attached to the surface of a dextran-coated liposome (DCL). IA-DCLs were characterized in vitro, and the pharmacokinetic and antitumor properties were assessed in vivo.The in vitro binding properties were measured with purified integrin, endothelial cells, and melanoma cells. The pharmacokinetic parameters were measured in healthy mice with 14C-labeled IA-DCLs and anti-tumor efficacy was assessed with the M21 human melanoma xenograft mouse model.In vitro, IC50 values for IA-DCLs and IA are similar, and IA-DCLs inhibit cell proliferation relative to controls. IA-DCLs are stable in serum, and the pharmacokinetic half-life in mice is 23 h. In the M21/mouse model, statistically significant inhibition of tumor growth was observed for mice treated with IA-DCLs, whereas controls including saline, DCLs lacking IA, and cyclo(RGDfV) were ineffective. Increased apoptosis and a reduction in vessel counts relative to controls were present in tumors from animals treated with IA-DCLs.These results demonstrate that IA-DCLs are potent anti-angiogenic therapeutic agents with superior in vivo activity and pharmacology compared to unmodified IA.

Tumor dependence on specific amino acids for survival and proliferation is well recognized and has been exploited effectively with the use of asparaginases for the treatment of acute lymphoblastic leukemia. Sensitivity of tumors to L-Arginine (L-Arg) deprivation results from an impaired ability to make L-Arg as a result of decreased functional expression of one or more of the three enzymes of the L-Arg biosynthetic pathway: ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS1) and argininosuccinate lyase (ASL). Native human arginase I is not a viable drug candidate due to low activity and low stability in serum. We have developed an alternative approach using a bioengineered human PEGylated arginase I (AEB1102) with enhanced pharmacological properties. We and others have successfully utilized arginase I to impart a direct tumor cell killing effect through L-Arg starvation in multiple tumor types e.g. AEB1102 single agent efficacy in melanoma, small cell lung cancer (SCLC) and sarcoma PDx models. However, the compatibility of AEB1102 with checkpoint inhibitors is unclear as arginase I has been shown to be both immune suppressive and immune neutral (PMID: 23717444), or immune promoting (PMID: 27043409). Because of these conflicting reports we decided to investigate the impact of systemic L-Arg removal on the anti-tumor efficacy of check point inhibitors. Murine syngeneic models (e.g. CT26, MC38 and LL2) were dosed with AEB1102 alone and in combination with anti-PD-L1 and anti-PD-1 monoclonal antibodies (mAbs). Depending on the model, in vivo treatment with AEB1102 monotherapy resulted in tumor growth inhibition (TGI) ranging from 52% to 72% compared to the untreated control group, whereas standard monotherapy using immunomodulatory mAbs that target PD-1 and PD-L1 resulted in TGI ranging from 12% to 60%. Of significance, combination therapy of AEB1102 with anti-PD-1 or anti-PD-L1 resulted in additive anti-tumor effect with TGI ranging from 60% to 86%. In the CT26 model, when AEB1102 was administered in combination with anti-PD-L1 for at least 6 weeks, a 33% frequency of complete tumor regression (non-palpable tumors) was observed, indicating that synergy occurs with combination therapy. Collectively these results demonstrate that disrupting the L-Arg physiological balance in the tumor microenvironment inhibits tumor growth and further sensitizes the tumor to immunotherapy when AEB1102 is combined with anti-PD-1 and anti-PD-L1. These data open the possibility of further improving outcomes in L-Arg dependent tumors through combination of AEB1102 with anti-PD-1 and anti-PD-L1 inhibitors. Citation Format: Giulia Agnello, Susan E. Alters, David G. Lowe, Scott W. Rowlinson. Reducing systemic arginine with arginase (AEB1102) therapy does not suppress the immune response induced by anti-PD-1 and anti-PD-L1, and exerts an additive anti-tumor and synergistic survival benefit [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 3964. doi:10.1158/1538-7445.AM2017-3964

Immunization with tumor-specific-associated antigen--pulsed dendritic cells has proved to be efficacious in various animal models and is being evaluated for the treatment of cancer in humans. Use of dendritic cells pulsed with specific peptides or transfected with tumor-associated antigen genes has been a focused area of investigation for inducing potent tumor and viral immune responses. In this study, the authors demonstrate transgene expression, including the lacZ and MART-1 genes, in dendritic cells infected with adenoviral constructs. These transiently transduced dendritic cells, derived from melanoma patients' monocytes cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4, express the transgene and can stimulate patients' CD8+ T cells to elicit an antitumor immune response comparable to dendritic cells loaded with a defined peptide. These cytotoxic T lymphocytes were able to recognize both known and unknown tumor-associated antigen epitopes and exhibited cytolytic activity against HLA-matched tumor cells expressing the antigen. The ability to induce tumor-specific cytotoxic T lymphocytes in vitro using gene-modified dendritic cells that transiently express tumor-associated antigens demonstrates the potential use of these antigen-presenting cells for developing in vivo cancer vaccines.

Cancer cells experience higher intrinsic oxidative stress than their normal counterparts and acquire adaptive antioxidant mechanisms to maintain redox balance. This increased antioxidant capacity has been correlated to malignant transformation, metastasis and resistance to standard anticancer drugs. This enhanced antioxidant state also correlates with cancer cells being more vulnerable to additional oxidative insults, therefore disruption of adaptive antioxidant mechanisms may have significant therapeutic implications. Hematological malignancies including Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM) are critically dependent on the cellular antioxidant glutathione (GSH), consistent with the higher intrinsic oxidative stress. L-cysteine is the rate-limiting substrate for GSH biosynthesis and adequate levels of cysteine are critical to maintain the intracellular homeostasis of GSH. CLL and a subset of ALL cells have been reported to rely on the stromal supply of cysteine to increase the synthesis of GSH in order to maintain redox balance, which in turn promotes cell survival and fosters drug resistance. One approach to target this cancer specific dependency is by therapeutic depletion of amino acids via enzyme administration; a clinically validated strategy for the treatment of ALL. Aeglea BioTherapeutics Inc. has developed a bioengineered cysteine and cystine degrading enzyme (Cyst(e)inase, AEB3103) and evaluated its therapeutic efficacy against hematological malignancies in in vitro, ex vivo and in vivo pre-clinical studies. The TCL1-TG:p53 -/- mouse model exhibits a drug resistant phenotype resembling human CLL with unfavorable cytogenetic alterations and highly aggressive disease progression. AEB3103 greatly decreased the viability of TCL1-TG:p53 -/- cells cultured in vitro, whereas the CLL therapeutic, fludarabine, showed minimal cytotoxic effects. In vivo treatment of TCL1-TG:p53 -/- mice with AEB3103 resulted in an increase in median survival time (7 months, p Disclosures Agnello: Aeglea BioTherapeutics: Employment. Alters:Aeglea BioTherapeutics: Employment, Equity Ownership. Tyler:Aeglea BioTherapeutics: Employment, Equity Ownership. Huang:Aeglea BioTherapeutics: Research Funding. Stone:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Research Funding; University of Texas at Austin: Employment, Patents & Royalties: I am an inventor of technology related to this abstract. Georgiou:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Lowe:Aeglea BioTherapeutics: Employment, Equity Ownership. Rowlinson:Aeglea BioTherapeutics: Employment, Equity Ownership.

Introduction: Tumor dependence on specific amino acids for survival and proliferation is well recognized and has been exploited effectively with the use of Asparaginase for the treatment of Acute Lymphoblastic Leukemia. Decades of research have led to an understanding of tumor L-Arginine (L-Arg) dependence, with functional expression of the three enzymes of the L-Arg biosynthetic pathway: Ornithine Transcarbamylase (OTC), Argininosuccinate Synthase (ASS1) and Argininosuccinate Lyase (ASL) being required to convert ornithine to L-Arg. In a variety of tumor types, silencing of one or more of these enzymes disables endogenous L-Arg synthesis forcing a reliance on the extracellular pool of L-Arg for tumor survival and proliferation. This mechanism has been confirmed with Arginine Deiminase (ADI-PEG) and pegylated wild-type Arginase I (BCT-100). Aeglea Biotherapeutics Inc. has developed an alternative approach using a bioengineered human PEGylated Arginase I with enhanced pharmacological properties. Replacement of manganese, the natural metal co-factor in wild-type human Arginase I, with cobalt confers improved catalytic activity and serum stability. The resulting product candidate (AEB1102) displays highly favorable PK/PD properties, and is expected to be naturally tolerated by the human immune system as no protein modifications have been introduced. Experimental Procedures and Results: Non-clinical dose range finding and GLP toxicology studies were performed with AEB1102 in monkeys and mice. Bioanalytical assays detecting L-Arg and AEB1102 enzyme activity were used to monitor PD/PK in the dose range finding studies with these results subsequently being used to design the toxicology studies. Subsequent toxicology studies identified an NOAEL in both species at a dose that is predicted to translate to significant sustained reduction of L-Arg serum levels with weekly intravenous dosing. To determine tumor types most likely to respond to AEB1102 expression profiling of OTC, ASS1 and ASL was performed using in situ hybridization on multiple tumor histologies. Melanoma was identified as a tumor type likely to respond to AEB1102 owing to a significant reduction in ASS1 expression. Non-clinical in vivo studies using the A375 melanoma xenograft as well as melanoma PDx models confirm sensitivity to AEB1102, weekly dosing resulted in a significant delay in tumor growth and improved survival. Conclusion: Non-clinical activities required to support the IND submission of AEB1102 for solid tumors were successfully executed, enabling the Phase 1 study to be initiated in October 2015. Translational work profiling the expression of OTC, ASS1 and ASL has identified melanoma as a relevant tumor type to pursue in future clinical studies. Citation Format: Scott W. Rowlinson, Susan E. Alters, Giulia Agnello, Joseph Tyler, Ann Lowe, Mauri Okamoto-Kearney, Dale Johnson, Everett Stone, George Georgiou, David G. Lowe. Development of AEB1102, an engineered human arginase 1 for patients with solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1042.

Objectives Glucagon-like peptide 2 (GLP2) is an intestinal growth factor that has been shown to stimulate intestinal growth and reduce disease severity in preclinical models of short bowel syndrome and inflammatory bowel disease. Teduglutide, a recombinant human GLP2 variant (GLP2-2G), has increased half-life and stability as compared to the native GLP2 peptide, but still requires twice daily dosing in preclinical models and daily dosing in the clinic. The goal of this study was to produce and characterize the preclinical pharmacokinetic and therapeutic properties of GLP2-2G-XTEN, a novel, long-acting form of GLP2-2G. Methodology and Results A GLP2-2G-XTEN fusion protein with extended exposure profile was produced by genetic fusion of GLP2-2G peptide to XTEN, a long, unstructured, non-repetitive, hydrophilic sequence of amino acids. The serum half-life of GLP2-2G-XTEN in mice, rats and monkeys was 34, 38 and 120 hours, respectively. Intestinotrophic effects were demonstrated in normal rats, where GLP2-2G-XTEN administration resulted in a significant increase in both small intestine weight and length. Efficacy of the GLP2-2G-XTEN protein was compared to that of GLP2-2G peptide in a rat Crohn’s disease model, indomethacin-induced inflammation. Prophylactic administration of GLP2-2G-XTEN significantly increased the length, reduced the number of trans-ulcerations and adhesions, and reduced the TNFα content of the small intestine. GLP2-2G-XTEN demonstrated greater in vivo potency as compared to GLP2-2G peptide, and improvement in histopathology supported the GLP2-2G-XTEN treatment effects. Conclusions and Significance GLP2-2G-XTEN is intestinotrophic and demonstrates efficacy in a rat Crohn’s disease model requiring a lower molar dose and less frequent dosing relative to GLP2-2G peptide. Allometric scaling based on pharmacokinetics from mouse, rat and monkey projects a human half-life of 240 hours. These improvements in preclinical pharmacokinetics and dosing indicate that GLP2-2G-XTEN may offer a superior therapeutic benefit for treatment of gastrointestinal diseases including Crohn’s disease.

Summary: Immunization with tumor-associated antigen pulsed dendritic cells (DC) has been shown to elicit both protective and therapeutic antitumor immunity in a variety of animal models and is currently being investigated for the treatment of cancer patients in clinical trials. In this study we show that DC can be generated from peripheral blood mononuclear cells of healthy donors as well as breast and melanoma cancer patients using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-13 (IL-13) and that these DC have many of the same characteristics as DC differentiated using GM-CSF and IL-4. The DC generated in GM-CSF and IL-13 are CD 14¯ and express high levels of the cell surface markers CD86, HLA-DR, and CD58, as do DC generated in GM-CSF and IL-4. The purity and yield of both DC populations are not significantly different. Furthermore, both populations of DC are effective at presentation of alloantigen as determined in a mixed lymphocyte response, and both are able to process and present soluble tetanus toxoid antigen to CD4+ T cells. Because we are interested in the generation of DC for antigen-specific cytotoxic T lymphocyte (CTL) generation, we compared the ability of peptide-pulsed DC differentiated in GM-CSF and IL-4 versus GM-CSF and IL-13 for the generation of influenza and MART-1 specific CTL. Both populations of DC induced CD3+CD8+CD4¯ and CD56¯ CTL, which could lyse the appropriate targets in an antigen-specific manner. Finally, both GM-CSF and IL-4 DC and GM-CSF and IL-13 DC yielded similar (3 galactosidase expression levels after transduction with recombinant adenovirus containing the LacZ gene. These results suggest that DC generated in GM-CSF and IL-13 may be useful for immunotherapy and gene therapy protocols.

The human carcinoembryonic antigen (CEA), which is expressed in several cancer types is a potential target for antigen-specific immunotherapy. In this study, we show that dendritic cells (DC) pulsed with an HLA class I restricted CEA cytotoxic T lymphocyte (CTL) peptide epitope can stimulate T cells to kill CEA peptide loaded T2 target cells as well as CEA expressing tumor lines in the presence of interleukin-7 (IL-7) in an HLA-restricted manner. This has been demonstrated for carcinoma patients as well as healthy donors. The DC-CEA + IL7 stimulated cultures contained predominantly CD3 + CD8 + CD56 - cells indicative of MHC class I resticted CTL. In addition, DC-CEA + IL-7 stimulated cells showed higher levels of CD69 expression compared with cells stimulated with IL-7 alone, implying an activated phenotype. When the T-cell receptor (TCR) from CTL cultures stimulated with DC-CEA + IL-7 was analyzed, an oligoclonal pattern of expression was found for certain Vβ subfamilies compared with the polyclonal patterns shown by IL-7 or phytohemagglutinin stimulated T cells from the same donors. This TCR restriction appeared to be maintained and enhanced after additional rounds of restimulation with DC-CEA + IL-7. The association between cytotoxicity and TCR restriction suggests that TCR analysis may be useful as an in vitro indicator to monitor alterations in the T-cell population in response to antigen-specific immunotherapies.

Antigen specific cytotoxic T lymphocytes (CTL) are being studied for their potential immunotherapeutic benefit in the treatment of cancer. Carcinoembryonic antigen (CEA) is an oncofetal protein best known for its overexpression in the majority of colorectal, gastric, pancreatic, non small cell lung, and breast carcinomas. We are using dendritic cells (DC) pulsed with the CEA CTL peptide epitope to generate CEA specific CTL. DC from HLA A2+ donors were isolated by culturing plastic adherent PBMC in GMCSF and 1L4 for 7 days. As expected these DC expressed the relevant cell surface molecules including HLA DR, CD58, CD80, and CD86. The DC were strippped of their endogenous peptides, pulsed with the A2 restricted CEA peptide, irradiated and used to stimulate autologous CD8+ T cells in the presence of 1L7. Using this approach we have been able to generate CEA specific CTL from the PBMC of breast and pancreatic carcinoma patients as well as normal donors. These CTL can lyse CEA peptide pulsed T2 targets as well as HLA A2+ tumor cells expressing the CEA antigen. This data is being used to support a phase I active immunotherapy clinical protocol using DC pulsed with CEA peptide to treat patients with metastatic malignancies expressing CEA.

Insulin dependent diabetes mellitus (IDDM) in the non-obese diabetic (NOD) mouse is the result of a cellular mediated autoimmune event that destroys pancreatic islet β cells. This destruction is characterized by a progressive lymphocytic infiltration into the islets as well as circulating autoantibodies and T cells reactive with islet antigens. To gain a better understanding of the cells responsible for islet destruction we isolated lymphocytes from the islets of prediabetic NOD mice and conducted a comparative phenotypic analysis with the analogous subpopulations of lymphocytes isolated from peripheral blood and lymph node (LN) of the same mice. CD3 + cells were analysed for T cell receptor (TcR); cell bearing γδ TcR were consistently observed at a higher frequency in the infiltrating T cells than in the periphery. Lymphocytes were also characterized for the expression of CD4 and CD8 T cell markers and, within each population, for the expression of activation markers (CD25, CD69) and adhesion markers (CD51, CD54, CD11b, CD49e, L-selectin). Significantly increased levels of CD4 + CD8 + double-positive and CD4 − CD8 − double-negative T cell populations were observed in the infiltrating lymphocytes as compared with peripheral lymphocytes. In addition, within both CD4 and CD8 subpopulations isolated from islet infiltrates, CD11b + and CD49e + cells were increased with respect to the same subset of cells isolated from the periphery. In contrast, the level of cells that expressed L-selectin was significantly higher in the periphery for both CD4 + and CD8 + cells than for infiltrating cells. These data describe the phenotype of islet reactive T cells in the NOD mouse and identify possible targets for therapeutic intervention.

Approximately 50% of patients with adult acute lymphoblastic leukemia (ALL) relapse after initial chemotherapy. The introduction of novel chemotherapeutics that target leukemia or leukemia-harboring tissues could improve response rates. AQ4N (banoxantrone; 1,4 bis[[2-(dimethylamino)ethyl]amino]-5,8-dihydroxyanthracene-9,10-dione bis N-oxide) is designed to have anti-tumor activity following a bioreduction step to AQ4, a DNA topoisomerase II inhibitor. Studies in rats and monkeys revealed that multiple doses of 10–20 mg/kg AQ4N were well tolerated. However, lymphopenia and atrophy of lymph nodes and spleen suggested selective activity in lymphoid cells. We postulated that AQ4N might have therapeutic potential in ALL. In a proliferation assay, using 3 previously established ALL cell lines, we compared in vitro anti-leukemic activity of AQ4N and AQ4 with daunorubicin, a commonly used component of induction therapy. AQ4 and daunorubicin displayed similar in vitro activity with IC50 values of 2–9 nM. AQ4N displayed little activity with IC50 values of 600–2,500nM. Subsequently, we evaluated in vivo efficacy of AQ4N in a xenotransplant model of human ALL. Eighteen NOD/scid mice were inoculated with primary human ALL cells. Leukemic cell counts (LCC) in peripheral blood were determined weekly. Engraftment was observed 23 days after inoculation and LCC increased exponentially. After 55 days mean LCC reached 0.5x106 cells/mL (range 0.1–0.9) and treatment was started. The cohort was randomized into 6 groups of 3 animals. Each group received three daily i.v. injections. Three groups received 3x20, 3x60 or 3x120 mg/kg AQ4N, respectively. Two groups received 3x2.0 or 3x5.0 mg/kg daunorubicin, respectively. Control mice received saline. Six days after the first injection, AQ4N and daunorubicin appeared equally effective. In the AQ4N treated groups mean LCC had decreased from 0.41, 0.48 and 0.55x106/mL to 0.016, 0.013 and 0.005x106/mL in the 20, 60 or 120 mg/kg treated groups (94.2, 97.3 and 99.2% reduction, respectively). In daunorubicin treated animals, mean LCC decreased from 0.15 and 0.17x106/mL to 0.014 and 0.004 x106/mL in the 2.0 or 5.0 mg/kg treated groups (90.6 and 97.2% reduction, respectively). Eleven days after the first administration, mean LCC remained at 0.01, 0.02 and 0.03x106/mL in the 20, 60 or 120 mg/kg AQ4N treated groups and at 0.02x106/mL in the group that received 5.0 mg/kg daunorubicin. However, in the group that received 2.0 mg/kg daunorubicin LCC had returned to pre-treatment values and leukemia progression was resumed. LCC in control treated animals increased throughout the study. In AQ4N treated animals a strong (90–99%) decrease of murine leukocyte counts occurred 6 to 11 days after treatment, supporting the hypothesis that AQ4N exerts specific activity in the leukocytic compartment. In contrast, in daunorubicin treated animals, murine leukocyte counts remained unaffected. The dose regimens used in this study were above the MTD in NOD/scid mice as toxicity was observed in all AQ4N and the 5.0 mg/kg daunorubicin treated groups. However, this systemic toxicity was a specific artefact for the NOD/scid strain as administration of three doses of 120 mg/kg AQ4N to BALB/c mice did not result in any toxicity. In conclusion, AQ4N may be a promising agent for the treatment of human ALL.