Preclinical Efficacy Studies Research Articles

Update on Standard Operating Procedures in Preclinical Research for DMD and SMA Report of TREAT-NMD Alliance Workshop, Schiphol Airport, 26 April 2015, The Netherlands.

A workshop took place in 2015 to follow up TREAT-NMD activities dedicated to improving quality in the preclinical phase of drug development for neuromuscular diseases. In particular, this workshop adressed necessary future steps regarding common standard experimental protocols and the issue of improving the translatability of preclinical efficacy studies.

An oral androgen receptor PROTAC degrader for prostate cancer.

381 Background: The Androgen Receptor (AR) remains the principal driver of castration-resistant prostate cancer during the transition from a localized to metastatic disease. Most patients initially respond to inhibitors of the AR pathway, but the response is often short-lived. The majority of patients progressing on enzalutamide or abiraterone exhibit genetic alterations in the AR locus, either in the form of amplifications or point mutations in the AR gene. Given these mechanisms of resistance, our goal is to eliminate the AR protein using the PROteolysis TArgeting Chimera (PROTAC) technology. Methods: Here we report an orally bioavailable small molecule AR PROTAC that leads to ubiquitination and degradation of AR. This molecule has been characterized in in vitro degradation and functional assays, DMPK, toxicology and preclinical efficacy studies. Results: This AR PROTAC completely degrades AR in all cell lines tested, with an observed 50% degradation concentration (DC50) < 1 nM. PROTAC-mediated AR degradation suppresses the expression of the AR-target gene PSA, inhibits AR-dependent cell proliferation, and induces potent apoptosis in VCaP cells. The AR PROTAC degrades all clinically relevant mutant AR proteins and retains activity in a high androgen environment. In mouse xenograft studies, greater than 90% AR degradation is observed at a 1 mg/kg PO QD dose. Significant inhibition of tumor growth and AR signaling can be achieved in both an intact and castrate setting. Further, the AR PROTAC demonstrates in vivo efficacy and reduction of oncogenic Erg protein in a long term, castrate, enzalutamide-resistant VCaP tumor model. DMPK and exploratory toxicology studies show robust oral, dose proportional drug exposure in rodent and non-rodent species. Conclusions: In summary, we report preclinical data on an orally bioavailable AR PROTAC degrader that demonstrates efficacy in enzalutamide-resistant prostate cancer.

Evaluation of the biological activities of the IL-15 superagonist complex, ALT-803, following intravenous versus subcutaneous administration in murine models

ALT-803 is a fusion protein complex consisting of an interleukin (IL)-15 superagonist and a dimeric IL-15 receptor alpha sushi domain IgG1 Fc fusion protein. When administered to mice, ALT-803 is capable of inducing natural killer (NK) and CD8+ T cell proliferation and activation, and effectively promoting potent anti-tumor responses. Currently, ALT-803 is in clinical trials for treatment of various solid tumors and hematological malignancies. In the initial phase of these clinical studies, intravenous (iv) injection was used according to the route used in pre-clinical efficacy studies. In order to evaluate the possible advantage of subcutaneous (sc) injection versus iv injection, this study compared the biological activity of the two treatment regimens of ALT-803 in pre-clinical in vivo models. The pharmacokinetics, immune stimulation, and anti-tumor efficacy of iv and sc injection routes of ALT-803 in C57BL/6 mice were compared. The half-life of ALT-803 was 7.5 h for iv versus 7.7 h for sc with the maximal detected serum concentration of ALT-803 to be 3926 ng/ml at 0.5 h time-point following iv injection versus 495 ng/ml at 16 h post sc injection. Biodistribution studies indicated that sc ALT-803, similarly to iv ALT-803 as previously reported, has a greater tissue distribution and longer residence time in lymphoid tissues compared to recombinant IL-15. Notably, ALT-803 when administered either iv or sc induced comparable proliferation and activation of CD8+ T and NK cells and resulted in similar reductions of tumor burden. A toxicity study of mice receiving multiple injections of ALT-803 for 4 weeks by iv or sc routes revealed equivalent immune-related changes. The gradual absorbance into the blood stream and lower maximal blood levels of ALT-803 in sc-injected mice, along with similar anti-tumor efficacy support the administration of ALT-803 by sc injection in patients with various malignancies and infectious diseases.

Establishing in vitro-in vivo correlation for antibody drug conjugate efficacy: a PK/PD modeling approach.

The objective of this manuscript was to establish in vitro-in vivo correlation (IVIVC) between the in vitro efficacy and in vivo efficacy of antibody drug conjugates (ADCs), using a PK/PD modeling approach. Nineteen different ADCs were used to develop IVIVC. In vitro efficacy of ADCs was evaluated using a kinetic cell cytotoxicity assay. The cytotoxicity data obtained from in vitro studies was characterized using a novel mathematical model, parameter estimates from which were used to derive an in vitro efficacy matrix for each ADC, termed as 'in vitro tumor static concentration' (TSCin vitro). TSCin vitro is a theoretical concentration at continuous exposure of which the number of cells will neither increase nor decrease, compared to the initial cell number in the experiment. The in vivo efficacy of ADCs was evaluated using tumor growth inhibition (TGI) studies performed on human tumor xenograft bearing mice. The TGI data obtained from in vivo studies was characterized using a PK/PD model, parameter estimates from which were used to derive an in vivo efficacy matrix for each ADC, termed as 'in vivo tumor static concentration' (TSCin vivo). TSCin vivo is a theoretical concentration if one were to maintain in the plasma of a tumor bearing mouse, the tumor volume will neither increase nor decrease compared to the initial tumor volume. Comparison of the TSCin vitro and TSCin vivo values from 19 ADCs provided a linear and positive IVIVC. The Spearman's rank correlation coefficient for TSCin vitro and TSCin vivo was found to be 0.82. On average TSCin vivo was found to be~27 times higher than TSCin vitro. The reasonable IVIVC for ADCs suggests that in vitro efficacy data was correctly able to differentiate ADCs for their in vivo efficacy. Thus, IVIVC can be used as a tool to triage ADC molecules in the discovery stage, thereby preventing unnecessary scaling-up of ADCs and waste of time and resources. An ability to predict the concentration of ADC that is efficacious in vivo using the in vitro data can also help in optimizing the experimental design of preclinical efficacy studies. As such, the novel PK/PD modeling method presented here to establish IVIVC for ADCs holds promise, and should be evaluated further using diverse set of cell lines and anticancer agents.

Discovery of a Cynomolgus Monkey Family With Retinitis Pigmentosa.

To accelerate the development of new therapies, an inherited retinal degeneration model in a nonhuman primate would be useful to confirm the efficacy in preclinical studies. In this study, we describe the discovery of retinitis pigmentosa in a cynomolgus monkey (Macaca fascicularis) pedigree. First, screening with fundus photography was performed on 1443 monkeys at the Tsukuba Primate Research Center. Ophthalmic examinations, such as indirect ophthalmoscopy, ERGs using RETeval, and optic coherent tomography (OCT) measurement, were then performed to confirm diagnosis. Retinal degeneration with cystoid macular edema was observed in both eyes of one 14-year-old female monkey. In her examinations, the full-field ERGs were nonrecordable and the outer layer of the retina in the parafoveal area was not visible on OCT imaging. Moreover, less frequent pigmentary retinal anomalies also were observed in her 3-year-old nephew. His full-field ERGs were almost nonrecordable and the outer layer was not visible in the peripheral retina. His father was her cousin (the son of her mother's older brother) and his mother was her younger half-sibling sister with a different father. The hereditary nature is highly probable (autosomal recessive inheritance suspected). However, whole-exome analysis performed identified no pathogenic mutations in these monkeys.

Abstract A185: Combining unbiased network analysis and cancer genomics to guide precision medicine-oriented early-phase clinical trials

Abstract Background: Precision medicine aims to tailor cancer therapies to target specific tumor-promoting aberrations. For tumors that lack actionable drivers, extensive molecular characterization and preclinical drug efficacy studies will be required to match a patient with the appropriate targeted therapy. Patients and Methods: Next-generation sequencing, high-throughput signaling network analysis, and drug efficacy trials were used in a tumor-derived cell line and a patient-derived xenograft (PDX) model to identify actionable targets for therapeutic intervention. Results: A patient-derived xenograft (PDX) model for preclinical research was successfully established and no actionable mutations were identified after whole exome sequencing of the patient’s DNA. Whole genome sequencing revealed amplification of the 3q and 5p chromosomal arms, which include the PIK3CA and RICTOR genes, respectively. Consistent with amplification of these genes, pathway analysis revealed activation of the AKT pathway. Based on this analysis, efficacy of PIK3CA and AKT inhibitors were evaluated, and response to the AKT inhibitor AZD5363 was observed in in vitro and in vivo models established from the patient’s biopsy, indicating the patient would benefit from targeted therapies directed against the serine/threonine kinase AKT. Conclusion: High-throughput signaling pathway analysis complements next-generation sequencing approaches for detection of actionable alterations and will aid in patient stratification into early-phase clinical trials. Citation Format: Pedro Torres-Ayuso, Sudhakar Sahoo, Christopher Chester, Cassandra Hodgkinson, Melanie Galvin, Hui Sun Leong, Kristopher Frese, Richard Marais, Caroline Dive, Matthew Krebs, John Brognard. Combining unbiased network analysis and cancer genomics to guide precision medicine-oriented early-phase clinical trials [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A185.

Animal welfare and corporate welfare in pharmaceutical R&D - the future of third-party assessment

For research and development (R&D) of new drugs, animal experimentation is indispensable, and research institutes, pharmaceutical companies, or contract research organizations routinely conduct preclinical studies of efficacy, safety, or metabolism using laboratory animals. However, animal experimentation entails some organizational risks. One is the suspension of R&D of a new drug, because in the course of clinical studies it becomes apparent that the drug has limited efficacy, unexpected side effects, and/or unexpected metabolites. Another risk is damage to the company image by development of an unfavorable reputation. Society has accepted animal experimentation as a necessary evil, but if such experimentation is not conducted with adequate concern for animal welfare, social sanctions will against that institute, company or organization will result. Once this happens, it is difficult to recover a good public image. Therefore, pharmaceutical companies must conduct animal experiments so as to obtain highly useful data without sacrificing public favor. One way to maintain a good reputation is through third-party accreditation, which verifies that the institute, company or organization and its researchers value animal welfare appropriately.

When Enough Is Enough: Decision Criteria for Moving a Known Drug into Clinical Testing for a New Indication in the Absence of Preclinical Efficacy Data.

Many animal models of disease are suboptimal in their representation of human diseases and lack of predictive power in the success of pivotal human trials. In the context of repurposing drugs with known human safety, it is sometimes appropriate to conduct the "last experiment first," that is, progressing directly to human investigations. However, there are not accepted criteria for when to proceed straight to humans to test a new indication. We propose a specific set of criteria to guide the decision-making around when to initiate human proof of principle without preclinical efficacy studies in animal models. This approach could accelerate the transition of novel therapeutic approaches to human applications.

Genetic testing in translational ophthalmology

Abstract Inherited eye diseases are a group of conditions with genetic and phenotypic heterogeneity. Advances in ocular genetic research have provided insights into the genetic basis of many eye diseases. Genetic and technological progress is improving the management and care of patients with inherited eye diseases. Diagnostic laboratories continue to develop strategies with high specificity and sensitivity that reduce the costs and time required for genetic testing. The introduction of next generation sequencing technologies has significantly advanced the identification of new gene candidates and has expanded the scope of genetic testing. Gene therapy offers an important opportunity to target causative genetic mutations. There are clinical trials of treatments involving vector-based eye gene therapies, and a significant number of loci and genes now have a role in the diagnosis and treatment of human eye diseases. Applied genetic technology heralds the development of individualized treatments, ushering ophthalmology into the field of personalized medicine. Many therapeutic strategies have demonstrated efficacy in preclinical studies and have entered the clinical trial phase. In this paper we review the topic of genetic testing in inherited eye diseases. We provide some background information about genetic counseling and genetic testing in ophthalmology and discuss how genetic testing can be helpful to patients and families with inherited eye diseases.

Metabolism, pharmacokinetics, and bioavailability of ZB716, a Steroidal Selective Estrogen Receptor Downregulator (SERD).

ZB716 is a selective estrogen receptor downregulator (SERD) with excellent oral bioavailability and superior efficacy. In this study, we investigate the in vitro and in vivo metabolism and the pharmacokinetics of ZB716 by incubation with liver microsomes, liver cytosol, and by orally dosing rodents. Metabolic products were identified and quantified by a combination of liquid chromatography and tandem +mass spectrometry. The metabolic profile of ZB716 showed fulvestrant and ZB716-sulfone as the two major oxidative metabolites. ZB716 also underwent some degree of sulfation and glucuronidation in vitro. The major oxidative metabolites of ZB716 were found in rat plasma, feces, and urine samples. No sulfation and glucuronidation metabolites from ZB716 were found in plasma. Limited amounts of sulfate conjugates and glucuronides of ZB716 were detected in feces. The glucuronidation on 3-OH position of fulvestrant was the main metabolite found in urine, suggesting that this specific site of phase 2 metabolism is blocked in ZB716 and formation of glucuronide 3-fulvestrant must be preceded by metabolic transformation of ZB716 to fulvestrant. The pharmacokinetic study of ZB716 showed a half-life (t1/2) at 17.03 hour, the area under curve value (AUC) of 1451.82 ng/ml*h, and the maximum plasma concentration (Cmax) at 158.12 ng/mL reached at 2 h after a single dose of 10 mg/kg by oral gavage. Overall this study elucidated important metabolic characteristics of ZB716, an oral SERD that has demonstrated superior bioavailability and efficacy in preclinical studies conducted so far.

Biochemical and aerosol characterization of ricin for use in non-clinical efficacy studies.

Ricin toxin may be used as a biological warfare agent and no medical countermeasures are currently available. Here, a well-characterized lot of ricin was aerosolized to determine the delivered dose for future pre-clinical efficacy studies. Mouse intraperitoneal (IP) median lethal dose (LD50 ) bioassay measured potency at 5.62 and 7.35μg/kg on Days 0 and 365, respectively. Additional analyses included total protein, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and rabbit reticulocyte lysate activity assay. The nebulizer aerosol produced consistent concentrations (2.5×103 , 5.0×103 , 1.0×104 , and 1.5×104 μg/mL) and spray factor values. The aerosol particle size distribution was of sufficient size to deposit in lung alveoli (1.12-1.43μm).Ricinus communis Agglutinin II (RCA 60), prepared at 19mg/mL in phosphate-buffered saline, pH 7.8, and stored at -70°C, maintained attributes for toxicity following 1-year storage and aerosolized consistently.

Natural Compounds for the Treatment of Psoriatic Arthritis: A Proposal Based on Multi-Targeted Osteoclastic Regulation and on a Preclinical Study.

BackgroundPsoriatic arthritis (PsA) is a chronic inflammatory arthritis affecting approximately 2% to 3% of the population globally, and is characterized by both peripheral articular manifestations and axial skeletal involvement. Conventional therapies for PsA have not been fully satisfactory, though natural products (NPs) have been shown to be highly effective and represent important treatment options for psoriasis. PsA is a multigenic autoimmune disease with both environmental and genetic factors contributing to its pathogenesis. Accordingly, it is likely that the use of natural compounds with a multi-targeted approach will enable us to develop better therapies for PsA and related disorders.ObjectivePsA, either on joint damage or on bone erosion, has been shown to respond to anti-psoriatic pharmacotherapy (APP), APP-like NPs, and their natural compounds. This study aims to uncover specific natural compounds for improved PsA remedies. Specifically, by targeting bone erosion caused by increased osteoclastic bone resorption, we aim to predict the key signaling pathways affected by natural compounds. Further, the study will explore their anti-arthritis effects using an in silico, in vitro, and in vivo approach. Following the signaling pathway prediction, a preclinical efficacy study on animal models will be undertaken. Collectively, this work will discover lead compounds with improved therapeutic effects on PsA.MethodsWe hypothesize that 9 potential APP-like NPs will have therapeutic effects on arthritis via the modulation of osteoclast bone resorption and signaling pathways. For in silico identification, the Latin name of each NP will be identified using the Encyclopedia of Traditional Chinese Medicine (Encyclopedia of TCM). The biological targets of NPs will be predicted or screened using the Herbal Ingredients' Targets (HIT) database. With the designed search terms, DrugBank will be used to further filter the above biological targets. Protein ANnotation THrough Evolutionary Relationship (PANTHER) will be used to predict the pathways of the natural compound sources. Subsequently, an in vitro sample preparation including extraction, fractionation, isolation, purification, and bioassays with high-speed counter-current chromatography-high-performance liquid chromatography-diode array detection (HSCCC-HPLC-DAD) will be carried out for each identified natural source. In vitro investigations into the effect of NPs on osteoclast signaling pathways will be performed. The experimental methods include cell viability assays, osteoclastogenesis and resorption pit assays, quantitative reverse transcription polymerase chain reaction (RT-PCR), western blot, and luciferase reporter gene assays. Finally, an in vivo preclinical efficacy on a collagen-induced arthritis rat model will be carried out using a treatment group (n=10), a control group (n=10), and a non-arthritis group (n=10). Main outcome measure assessments during intervention include daily macroscopic scores and a digital calipers measurement. Post-treatment tissue measurements will be analyzed by serological testing, radiographic imaging, and histopathological assessment.ResultsStudies are currently underway to evaluate the in silico data and the in vitro effects of compounds on osteoclastogenesis and bone resorption. The preclinical study is expected to start a year following completion of the in silico analysis.ConclusionsThe in silico rapid approach is proposed as a more general method for adding value to the results of a systematic review of NPs. More importantly, the proposed study builds on a multi-targeted approach for the identification of natural compounds for future drug discovery. This innovative approach is likely to be more precise, efficient, and compatible to identify the novel natural compounds for effective treatment of PsA.

Abstract 5130: Tumor-penetrating peptide-coated nanoparticles as a novel strategy for the targeted therapy of neuroblastoma

Abstract Anticancer drugs loaded into tumor- and vasculature-targeted nanocarriers (NC) can reduce side-effects and improve therapeutic efficacy in pre-clinical studies. However, poorly perfused and dysfunctional tumor vessels and lymphatics limit the transport of the payload into the parenchyma of solid tumors. The use of NC decorated with tumor-penetrating peptides (TPPs) might enhance tumor penetration and antitumor effects. A previously characterized neuroblastoma (NB)-targeting peptide ligand was here modified (now referred as TPP-NB) by adding a consensus motif as a mediator of cell, vascular and tissue penetration via neuropilin-1 (NRP-1) receptor recognition. NPR-1 expression was validated by FACS analysis in NB cell lines and by IHC staining in tumor cells and tumor stroma from NB-bearing mice. Recombinant NRP-1 was used to validate TPP-NB specificity. In vitro and in vivo cell association and internalization of TPP-NB, either free or coupled to Liposomes (L) were tested by FACS and confocal microscopy. Vascular permeability assay after treatment with TPP-NB-targeted, doxorubicin-loaded Liposomes (TPP-NB-L[DXR]) was performed evaluating the in vivo accumulation of Evans Blue dye within the tumor mass. Therapeutic experiments with TPP-NB-L[DXR] were performed in mice orthotopically injected with human NB cells. NRP-1 expression is validated in a panel of NB cells and in tumors from NB-bearing mice. Differently from the original peptide and some control ones, TPP-NB is able to recognize recombinant NRP-1. The addition of the NRP-1-recognizing sequence to the original peptide significantly increases its NB cellular association in vitro. Interestingly, the results seem to indicate that the enhanced capability by TPP-NB in binding NB cells is related to the combination of the NRP-1-recognizing and the original sequence. Importantly, TPP-NB coupled at the external surfaces of L[DXR] significantly increases their cellular association on NB cells in vitro. Competitive binding assay reveals that binding of TPP-NB is specific and can be inhibited by an excess of the unlabeled free peptide. The localization and the cellular distribution of L evaluated by confocal microscopy in vitro and in mouse models of NB, confirm the binding specificity, showing an increased selective internalization of TPP-NB-L-FITC compared to that obtained with either untargeted L or L decorated with the scrambled peptide. Moreover, TPP-NB-L[DXR] further increases the vascular permeability into the NB tumor mass, but not in non-tumor tissues. The therapeutic efficacy of TPP-NB-L[DXR] has been investigating in terms of overall survival. On running results indicate that the novel NC exerts an increased anti-NB effect compared to DXR-loaded L decorated with the original peptide. Our findings demonstrate that the achieved penetrating features by a NB-targeting peptide might increase liposomal drug binding, homing and antitumor efficacy. Citation Format: Fabio Pastorino, Chiara Brignole, Laura Emionite, Silvia Bruno, Flavio Curnis, Daniela Di Paolo, Patrizia Perri, Alessandro Gori, Renato Longhi, Michele Cilli, Angelo Corti, Mirco Ponzoni. Tumor-penetrating peptide-coated nanoparticles as a novel strategy for the targeted therapy of neuroblastoma [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 5130. doi:10.1158/1538-7445.AM2017-5130

Abstract 4568: A novel TGF-beta receptor I kinase inhibitor demonstrated to be an effective immuno-therapy for cancer

Abstract TGF-beta (TGF-β) pathways, despite serving as tumor suppressor in early stage of carcinogenesis, have shown be one of the key tumor-promoting factors in later-stage tumor progression. TGF-β receptor I kinase (TGF-βR1) inhibitors have shown great potentials in their anti-tumor and anti-metastasis efficacy in preclinical studies and some of them are being tested in the clinical settings. A specific TGF-βR1 inhibitor YL-13027 was developed at Yingli Pharmaceutical. YL-13027 possesses good ADME and PK properties and shows potent inhibition of the TGF-β and receptor interaction both in cell-free and cell-based assays. Functionally, in vitro Treg differentiation assay demonstrated the significant inhibition of the induction of FoxP3 expressing cells as well as inhibiting the secretion of IL-10 by YL-13027. In murine colon cancer CT26 model, YL-13027 was well tolerated and significantly inhibited tumor progression with dose dependency. More mechanistic studies are on-going. These data suggest that YL-13027, a novel TGF-βR1 inhibitor, represents a promising and safe immune modulator and shows great potential as a cancer immuno-therapeutics. Citation Format: Zusheng Xu, Yangtong Lou, Li Chen, Kun Zeng, Qingrui Sun, Yingying Qu, Wei Wang, Xiaodong Wu, Xiaomei Ge, Ying Gu, Yingjia Zhang, He Zhou. A novel TGF-beta receptor I kinase inhibitor demonstrated to be an effective immuno-therapy for cancer [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 4568. doi:10.1158/1538-7445.AM2017-4568

Abstract LB-288: An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED

Abstract Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12 and plays pivotal roles in transcriptional regulation through its histone H3K27 methyltransferase activity. Dysregulation of PRC2 is observed in multiple human cancers, for example, the catalytic subunit EZH2 is overexpressed in a wide range of human cancers and gain-of-function mutations of EZH2 within its catalytic site have been reported in human B-cell lymphoma, parathyroid carcinoma and melanoma. Small molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported and showed anti-lymphoma efficacy in pre-clinical studies. EED within the PRC2 complex allosterically activate the enzymatic activity by binding to tri-methylated H3K27 (H3K27me3). Here we report the discovery of EED226, a potent and selective PRC2 inhibitor directly binding to the H3K27me3 binding pocket of EED. EED226 induces conformational change upon binding EED leading to loss of PRC2 activity. EED226 shows similar activity as SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show EED226 inhibits PRC2 activity via an allosteric mechanism and offers opportunity for treatment of PRC2-dependent cancers. Citation Format: Wei Qi, Kehao Zhao, Justin Gu, Ying Huang, Youzhen Wang, Hailong Zhang, Man Zhang, Jeff Zhang, Zhengtian Yu, Ling Li, Lin Teng, Shannon Chuai, Chao Zhang, Mengxi Zhao, HoMan Chan, Zijun Chen, Douglas Fang, Fei Qi, Leying Feng, Lijian Feng, Yuan Gao, Hui Ge, Xinjian Ge, Andreas Lingel, Guobin Li, Ying Lin, Yueqin Liu, Fangjun Luo, Minlong Shi, Long Wang, Zhaofu Wang, Yanyan Yu, Jue Zeng, Chenhui Zeng, Lijun Zhang, Qiong Zhang, Shaolian Zhou, Counde Oyang, Peter Atadja, En Li. An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED [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 LB-288. doi:10.1158/1538-7445.AM2017-LB-288

Abstract 4600: Potent and selective C-C chemokine receptor (CCR4) antagonists potentiate anti-tumor immune responses by inhibiting regulatory T cells (Treg)

Abstract Naturally suppressive CD4+ Foxp3+ Treg are essential for immune tolerance. Although Treg-mediated suppression of effector cells is important to control inflammation and prevent autoimmune diseases, the presence of Treg in the tumor microenvironment (TME) has been shown to dampen anti-tumor immune responses. Human Treg express CCR4, the receptor for the chemokines CCL17 and CCL22. These chemokines are produced by tumor cells, tumor-associated macrophages and dendritic cells, as well as by effector T cells (Teff). Preclinical and clinical data supports a role for CCR4-mediated recruitment and accumulation of Treg in the TME which can be associated with poor prognosis. Further, recent longitudinal studies in patients receiving IO agents demonstrate an influx of Treg in responding patients which may dampen optimal anti-tumor responses. Therefore, CCR4 is an ideal target to selectively block Treg recruitment into the TME. We have developed structurally unique series of small molecule antagonists of CCR4. These antagonists have cellular potencies in multiple assays (e.g. chemotaxis of primary human Treg in 100% serum) in the low double-digit nM range. Representative compounds are selective against other chemokine receptors, GPCRs and ion channels, including the hERG channel, and lack inhibition of common human CYP450 enzymes. Moreover, compounds have excellent in vitro and in vivo ADME properties, consistent with convenient oral dosing. In preclinical syngeneic tumor models, these CCR4 antagonists block Treg migration and support expansion of activated Teff. In contrast to the non-selective approach of depleting anti-CCR4 antibodies, our compounds reduce Treg in the tumor, but not in peripheral tissues such as blood, spleen or skin. In preclinical efficacy studies, CCR4 antagonists potentiate the anti-tumor effects of various checkpoint inhibitors and immune stimulators such as anti-PD-L1 and anti-CD137 antibodies. We observe enhanced tumor growth inhibition and increased tumor regressions when these agents are combined with CCR4 antagonists, without any gross toxicity. Further characterization of these CCR4 antagonists and their anti-tumor effects will be described. Citation Format: Oezcan Talay, Lisa Marshall, Cesar Meleza, Maureen K. Reilly, Omar Robles, Mikhail Zibisky, Abood Okal, Lisa Seitz, Jenny McKinnell, Scott Jacobson, Erin Riegler, Emily Karbaz, David Chian, Angela Wadsworth, Paul Kassner, David Wustrow, Jordan S. Fridman. Potent and selective C-C chemokine receptor (CCR4) antagonists potentiate anti-tumor immune responses by inhibiting regulatory T cells (Treg) [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 4600. doi:10.1158/1538-7445.AM2017-4600

Abstract 4565: Forced expression of GITRL in cancer cells enhances adenovirus-mediated in situ vaccination

Abstract Our approach involves a platform of killing cancer using more potent oncolytic viruses-based immunotherapy strategies. These replication competent adenoviruses are targeted to the Rb pathway to generate tumor-selectivity. The second generation of these therapeutic agents, Delta-24-RGD, was successfully translated to the clinical setting and is currently been tested in Phase I studies in several institutions in the USA and in Europe for the treatment of patients suffering from recurrent gliomas. Preliminary data from these clinical trials showed that 10 to 15% of Delta-24-RGD-treated tumors undergo complete regression. Agonistic treatments targeting co-stimulatory tumor necrosis factor receptor superfamily (TNFRSF), such as GITR (CD357), have been shown to enhance the proliferation and activation of T cells. Moreover, in preclinical tumor efficacy studies, these agonistic signals have shown potent tumoricidal activity. Different from antibodies, co-stimulatory ligands can be easily incorporated into replication competent oncolytic adenoviruses. Infection of cancer cells with these armed viruses will lead to the expression in their cell membranes of the co-stimulatory molecule that will directly interact with the tumor infiltrating lymphocytes to amplify and enhance the anti-tumor T-cell activity. In this study, we have developed an armed Delta-24-RGD carrying the cDNA of the mouse GITRL, Delta-24-GREAT. Treatment of glioma-bearing mice with intracranial injection of Delta-24-GREAT increased mice survival (P < 0.0001, long-rank test) and inhibited lung cancer growth in subcutaneous models. Infection of the tumor elicited an inflammatory response increasing the populations of CD4+ and CD8+ T cells versus treated controls. Importantly, 2 weeks after the adenovirus treatment a subset of brain hemispheric cells were positive for GITRL. In addition, co-culture experiments with tumor cells infected with viruses and splenocytes isolated from treated glioma-bearing mice demonstrated a response against the cancer cells as assessed by ELISA analyses of IFN-γ. Demonstrating the generation of anti-tumor memory, the surviving animals did not show evidence of tumor growth after re-challenging with GL261 glioma cell implantation in the contralateral hemisphere. However, survivors of GL261 tumors did not survive after the re-challenge was performed by intracranial implantation of B16/F10 melanoma cells, strongly indicating that the immune response was specific for GL261 glioma antigens. This is the first study with an oncolytic adenovirus expressing GITRL and our results strongly indicate that oncolytic adenoviruses armed with molecules of the TNFRSF may be of future clinical interest for the treatment of patients with cancer. Citation Format: Yisel A. Rivera-Molina, Francisco Puerta Martinez, Teresa Nguyen, Hong Jiang, Xuejun Fan, Rehnuma Shifat, Mohammad Belayat Hossain, Verlene K. Henry, Caroline C. Carrillo, Candelaria Gomez-Manzano, Juan Fueyo. Forced expression of GITRL in cancer cells enhances adenovirus-mediated in situ vaccination [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 4565. doi:10.1158/1538-7445.AM2017-4565

Abstract 2805: A panel of orthotopic glioblastoma multiforme (GBM) patient derived xenograft (PDX) mouse models for efficacy evaluation of drugs

Abstract Background: Patients with glioblastoma multiforme (GBM) have a poor prognosis and few treatment options; hence new treatments are needed. Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, orthotopic implantation confers a translational advantage as the cancer develops in a microenvironment more closely mimicking that of the original patient tumor. Also the major impact of the blood brain barrier that must be taking into account when targeting brain tumors as GBM in terms of drug bioavailability is better represented in the orthotopic models. The aim of this study was therefore to develop a panel of orthotopic GBM PDX models for pre-clinical efficacy studies of new drugs. The models were then used to study the efficacy of standard of care such as temozolomide (TMZ) and external radiation therapy (XRT). Methods: Low passage subcutaneous tumors from six different PDX GBM models designated ST108, ST112, ST146, ST545, ST610 and ST2473 were digested and used for intracranial stereotactic injection in nude mice. Tumor take and growth was determined by T2-weighted magnetic resonance imaging (MRI). At confirmed tumor take mice were either treated with TMZ (100mg/kg/day for 5 days) or whole brain XRT (2 Gy/day for 5 days). Control groups receiving vehicle or sham XRT were included depending on treatment regiment. Final endpoint was survival by humane endpoints and tumors were fixed in formalin for histological evaluation. Results: MRI confirmed tumor take in all models within 5 weeks of implantation. The take rate was > 80% across all models. TMZ showed efficacy in the orthotopic ST610 GBM PDX model evaluated by MRI on day 14 (16.2±2.9 mm3 vs. 76.8±13.1 mm3, p=0.016), whereas the ST146 model displayed resistance to TMZ on day 14 (12.7±5.6 mm3 vs. 26.5±11.9 mm3, p=0.26). The median survival was 60 days vs. 14 days in the ST610 model (TMZ vs. vehicle, p=0.0005) and 27 days vs. 13 days in the ST146 model (TMZ vs. vehicle, p=0.007). XRT showed efficacy in the orthotopic ST2473 model. Tumor volume was significantly smaller in treated vs. sham animals 11 days after inclusion (6.9±1.4 mm3 vs. 28.9±3.3 mm3, p=0.001). Also, a survival benefit was observed in XRT treated animals compared to sham. Histology confirmed the presence of orthotopic tumors and typical GBM pathology characteristics such as pseudopalisading tumor cells surrounding necrosis and micro vascular proliferation were identified. Conclusion: Six different orthotopic GBM PDX models were established from low passage subcutaneous PDX models. Models sensitive and resistant to TMZ were identified and histological GBM characteristics were identified. Together, the established panel of orthotopic PDX models can be used as a relevant translational platform for testing of new drugs in a setting that more closely mimics the GBM tumor microenvironment and the impact of the blood brain barrier in patients. Citation Format: Mette M. Jensen, Camilla S. Knudsen, Lotte K. Kristensen, Mette K. Nedergaard, Michael J. Wick, Kyriakos P. Papadopoulos, Anthony W. Tolcher, Andreas Kjaer, Carsten H. Nielsen. A panel of orthotopic glioblastoma multiforme (GBM) patient derived xenograft (PDX) mouse models for efficacy evaluation of drugs [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 2805. doi:10.1158/1538-7445.AM2017-2805

ONC201 selectively induces apoptosis in cutaneous T-cell lymphoma cells via activating pro-apoptotic integrated stress response and inactivating JAK/STAT and NF-κB pathways.

Cutaneous T-cell lymphomas (CTCLs) are extremely symptomatic and still incurable, and more effective and less toxic therapies are urgently needed. ONC201, an imipridone compound, has shown efficacy in pre-clinical studies in multiple advanced cancers. This study was to evaluate the anti-tumor activity of ONC201 on CTCL cells. The effect of ONC201 on the cell growth and apoptosis were evaluated in CTCL cell lines (n=8) and primary CD4+ malignant T cells isolated from CTCL patients (n=5). ONC201 showed a time-dependent cell growth inhibition in all treated cell lines with a concentration range of 1.25-10.0 μM. ONC201 also induced apoptosis in tested cells with a narrow concentration range of 2.5-10.0 μM, evidenced by increased Annexin V+ cells, accompanied by accumulated sub-G1 portions. ONC201 only induced apoptosis in CD4+ malignant T cells, not in normal CD4+ T cells. The activating transcription factor 4 (ATF4), a hallmark of integrated stress response, was upregulated in response to ONC201 whereas Akt was downregulated. In addition, molecules in JAK/STAT and NF-κB pathways, as well as IL-32β, were downregulated following ONC201 treatment. Thus, ONC201 exerts a potent and selective anti-tumor effect on CTCL cells. Its efficacy may involve activating integrated stress response through ATF4 and inactivating JAK/STAT and NF-κB pathways.

DNA vaccination protects mice against Zika virus-induced damage to the testes

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract.