Susan E. Alters

A novel long‐acting human growth hormone fusion protein (vrs‐317): enhanced in vivo potency and half‐life

A novel recombinant human growth hormone (rhGH) fusion protein (VRS-317) was designed to minimize receptor-mediated clearance through a reduction in receptor binding 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 threefold 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 1.4 mg/kg VRS-317 (equivalent to 0.26 mg/kg rhGH) caused a sustained pharmacodynamic response for 1 month equivalent to 0.05 mg/kg/day rhGH (1.4 mg/kg rhGH total over 28 days). In monkeys, VRS-317, having a terminal elimination half-life of approximately 110 h, 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 observations in these animal studies.

Selective Tumor Targeting by the Hypoxia-Activated Prodrug AQ4N Blocks Tumor Growth and Metastasis in Preclinical Models of Pancreatic Cancer

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.

Enhancement of the efficacy of an antagonist of an extracellular receptor by attachment to the surface of a biocompatible carrier.

AbstractPurpose. In order to improve the in vitro and in vivo efficacy of an integrin antagonist (IA) of the extracellular domain of the αvβ3 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 anti-tumor properties were assessed in vivo. Methods. 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. Results.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. Conclusions. These results demonstrate that IA-DCLs are potent anti-angiogenic therapeutic agents with superior in vivo activity and pharmacology compared to unmodified IA.

Abstract 1042: Development of AEB1102, an engineered human arginase 1 for patients with solid tumors

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.

Abstract 3964: 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

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

GLP2-2G-XTEN: a pharmaceutical protein with improved serum half-life and efficacy in a rat Crohn's. PLoS One. 2012; 7(11): e50630.

[This corrects the article on p. e50630 in vol. 7.].