Supp Figures S1 to S6 and Supp Tables S1 to S2

Objective While microCT evaluation of atherosclerotic lesions in mice has been formally validated, existing image processing methods remain undisclosed. We aimed to develop and validate a reproducible image processing workflow based on phosphotungstic acid-enhanced microCT scans for the volumetric quantification of atherosclerotic lesions in entire mouse aortas. Approach and Results. 42 WT and 42 apolipoprotein E knockout mouse aortas were scanned. The walls, lumen, and plaque objects were segmented using dual-threshold algorithms. Aortic and plaque volumes were computed by voxel counting and lesion surface by triangulation. The results were validated against manual and histological evaluations. Knockout mice had a significant increase in plaque volume compared to wild types with a plaque to aorta volume ratio of 0.3%, 2.8%, and 9.8% at weeks 13, 18, and 26, respectively. Automatic segmentation correlated with manual (r2 ≥ 0.89; p < .001) and histological evaluations (r2 > 0.96; p < .001). Conclusions The semiautomatic workflow enabled rapid quantification of atherosclerotic plaques in mice with minimal manual work.

Cancer vaccines using synthetic long peptides (SLP) targeting tumor antigens have been tested in the clinic but the outcomes have been unimpressive, perhaps because these peptides elicit predominantly CD4 + T cell responses. We hypothesized that enhanced delivery of peptide antigens to, and uptake in, secondary lymphoid tissues should elicit more robust CD8 + and CD4 + T cell responses and improved anti-tumor responses. Here, we have designed SLP-containing cationic lipoplexes (SLP–Lpx) that improve delivery of peptides to myeloid cells in the spleen and lymphatics. Using the G12D KRAS mutations as neoantigens, we found that vaccination of mice with naked synthetic peptides harboring the G12D mutation with CpG adjuvant stimulated mainly CD4 + T cell responses with limited tumor growth inhibition. On the other hand, immunization with SLP–Lpx stimulated both CD4 + and CD8 + T cells and suppressed tumor growth in a CD8 + T cell-dependent manner. Combination of the SLP–Lpx vaccines with a checkpoint inhibitor led to profound growth suppression of established tumors. These studies suggest that preferential targeting of peptides derived from neoantigens to the spleen via lipoplexes elicits potent CD4 + and CD8 + T cell responses that inhibit tumor growth.

Romosozumab (EVENITY™ [romosozumab-aqqg in the US]) is a humanized monoclonal antibody that inhibits sclerostin and has been approved in several countries for the treatment of osteoporosis in postmenopausal women at high risk of fracture. Sclerostin is expressed in bone and aortic vascular smooth muscle (AVSM). Its function in AVSM is unclear but it has been proposed to inhibit vascular calcification, atheroprogression, and inflammation. An increased incidence of positively adjudicated serious cardiovascular adverse events driven by an increase in myocardial infarction and stroke was observed in romosozumab-treated subjects in a clinical trial comparing alendronate with romosozumab (ARCH; NCT01631214) but not in a placebo-controlled trial (FRAME; NCT01575834). To investigate the effects of sclerostin inhibition with sclerostin antibody on the cardiovascular system, a comprehensive nonclinical toxicology package with additional cardiovascular studies was conducted. Although pharmacodynamic effects were observed in the bone, there were no functional, morphological, or transcriptional effects on the cardiovascular system in animal models in the presence or absence of atherosclerosis. These nonclinical studies did not identify evidence that proves the association between sclerostin inhibition and adverse cardiovascular function, increased cardiovascular calcification, and atheroprogression.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need for chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analoging. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in linker, peptide loading, and antibody attachment site. We found conjugates with 100x improved in vitro potency relative to complementary GpTx-1 analogs, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold whilst retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analog with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss in peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.

The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described. A cryopreserved plated mouse hepatocyte assay was designed to measure the depletion of the peptide-antibody conjugates from the media, with a correlation being observed between percentage remaining in the media and in vivo clearance (Pearson r = -0.5525). Physicochemical (charge and hydrophobicity), receptor-binding [neonatal Fc receptor (FcRn)], and in vivo pharmacokinetic data were generated and compared with the results from our in vitro hepatocyte assay, which was hypothesized to encompass all of the aforementioned properties. Correlations were observed among hydrophobicity; FcRn binding; depletion rates from the hepatocyte assay; and ultimately, in vivo clearance. Subsequent studies identified potential roles for the low-density lipoprotein and mannose/galactose receptors in the association of the Nav1.7 peptide conjugates with mouse hepatocytes, although in vivo studies suggested that FcRn was still the primary receptor involved in determining the pharmacokinetics of the peptide conjugates. Ultimately, the use of the cryopreserved hepatocyte assay along with FcRn binding and hydrophobic interaction chromatography provided an efficient and integrated approach to rapidly triage molecules for advancement while reducing the number of in vivo pharmacokinetic studies. SIGNIFICANCE STATEMENT: Although multiple in vitro and in silico tools are available in small-molecule drug discovery, pharmacokinetic characterization of protein therapeutics is still highly dependent upon the use of in vivo studies in preclinical species. The current work demonstrates the combined use of cryopreserved hepatocytes, hydrophobic interaction chromatography, and neonatal Fc receptor binding to characterize a series of Nav1.7 peptide-antibody conjugates prior to conducting in vivo studies, thus providing a means to rapidly evaluate novel protein therapeutic platforms while concomitantly reducing the number of in vivo studies conducted in preclinical species.

Abstract Purpose: Talimogene laherparepvec, a new oncolytic immunotherapy, has been recently approved for the treatment of melanoma. Using a murine version of the virus, we characterized local and systemic antitumor immune responses driving efficacy in murine syngeneic models. Experimental Design: The activity of talimogene laherparepvec was characterized against melanoma cell lines using an in vitro viability assay. Efficacy of OncoVEXmGM-CSF (talimogene laherparepvec with the mouse granulocyte-macrophage colony-stimulating factor transgene) alone or in combination with checkpoint blockade was characterized in A20 and CT-26 contralateral murine tumor models. CD8+ depletion, adoptive T-cell transfers, and Enzyme-Linked ImmunoSpot assays were used to study the mechanism of action (MOA) of systemic immune responses. Results: Treatment with OncoVEXmGM-CSF cured all injected A20 tumors and half of contralateral tumors. Viral presence was limited to injected tumors and was not responsible for systemic efficacy. A significant increase in T cells (CD3+/CD8+) was observed in injected and contralateral tumors at 168 hours. Ex vivo analyses showed these cytotoxic T lymphocytes were tumor-specific. Increased neutrophils, monocytes, and chemokines were observed in injected tumors only. Importantly, depletion of CD8+ T cells abolished all systemic efficacy and significantly decreased local efficacy. In addition, immune cell transfer from OncoVEXmGM-CSF-cured mice significantly protected from tumor challenge. Finally, combination of OncoVEXmGM-CSF and checkpoint blockade resulted in increased tumor-specific CD8+ anti-AH1 T cells and systemic efficacy. Conclusions: The data support a dual MOA for OncoVEXmGM-CSF that involves direct oncolysis of injected tumors and activation of a CD8+-dependent systemic response that clears injected and contralateral tumors when combined with checkpoint inhibition. Clin Cancer Res; 23(20); 6190–202. ©2017 AACR.

496 Objectives The noninvasive detection and quantification of T cells is important for monitoring success of immuno-therapeutics in the fields of oncology, autoimmunity, and infection.1 Current methods for lymphocyte detection are limited to cell isolation from peripheral blood; due to the invasive nature of tissue biopsy such sampling is done less frequently.2 Thus, methods for dynamic measuring of T cell localization and migration beyond peripheral blood in vivo remains technically challenging. The objective of this study was to develop a PET probe for imaging the endogenous distribution of T cells in murine models. Methods Parental 2C11 anti-mouse CD3 antibody was enzymatically fragmented with pepsin.3 The generated F(ab’)2 fragment was modified with NOTA-NCS via non-specific lysine conjugation. Tracer binding affinity was determined via a competitive inhibition assay on isolated murine T cells.4 The biodistribution of the tracer was evaluated in immune compromised NSG and competent, Balb/c mice at 4 and 24 h p.i. Results Pepsin digestion effectively removed the Fc region of the antibody, the resulting F(ab’)2 fragment was isolated with a product yield of 31 %. Optimal Cu-64 labeling was achieved with 0.1 M NH4OAc pH 5.5, with a specific activity of 2 Ci/µmol. A three-fold decrease in CD3 binding affinity was observed as a result of fragmentation and NOTA loading [IgG Kd = 13.7; F(ab’)2-NOTA Kd = 41.3 nM]. In immune competent mice, the tracer specifically accumulated in lymphoid organs (lymph nodes and spleen). Conversely, in immune compromised NSG mice accumulation was limited to the kidneys due to tracer clearance. Conclusions The anti-mouse CD3 F(ab’)2 PET probe was successfully used to image the endogenous distribution of T cells in vivo. The development of this tracer can potentially serve as a noninvasive tool for the dynamic tracking of T cell migration in syngeneic immunotherapy models. Research Support Amgen Inc.

Meeting abstracts Talimogene laherparepvec (T-VEC) is an injectable modified oncolytic herpes simplex virus type-1 (HSV-1) hypothesized to be efficacious by at least two complimentary mechanisms of action: a) direct oncolysis of the injected tumor and b) elicitation of a systemic anti-tumor immune

1809 Objectives Inhibitors of PDE10A, a phosphodiesterase expressed in the GABAergic neurons of the striatum, are efficacious in rodent models of schizophrenia and have potential benefit in multiple neurological disorders. A novel PET tracer was used to determine target coverage and binding characteristics of a PDE10A inhibitor in the rat brain. Methods SD rats were administered the PDE10A inhibitor MP-10 or vehicle 110 min prior to PDE10A tracer injection, followed by 120 min dynamic PET scans. T2-weighted MRI was performed on one animal to generate an anatomic brain template that was registered to the PDE10A tracer PET scan of the same animal. A volume of interest (VOI) set of brain regions was defined on the MRI template scan. Tracer uptake was quantified using template-based VOI analysis: the PDE10A PET scan of the template animal was affine-registered to individual PDE10A PET tracer scans, and the VOI set was mapped to FDG PET images to determine regional uptakes. Standardized uptake value (SUV) and binding potential (BP) were calculated for each region. Results In vehicle-treated animals, pronounced tracer uptake was observed in the PDE10A-abundant striatum (SUV=3.0±0.4; BP=3.4±0.4) while uptake in the prefrontal cortex, hippocampus, and cerebellum was similar to background (SUV≈0.6; BP≈0). In animals treated with the PDE10A inhibitor MP-10, tracer uptake was significantly reduced in the striatum (SUV=1.9±0.1, 36% decrease; BP=1.4±0.1, 59% decrease) while SUV and BP in the prefrontal cortex, hippocampus, and cerebellum were comparable to vehicle-treated animals. Conclusions The PDE10A PET tracer distributed to brain regions consistent with PDE10A expression and exhibited on-target binding as measured by competitive inhibition with MP-10. This novel PDE10A tracer provides a noninvasive method to measure target coverage and could provide utility in lead optimization and human dose selection.

Abstract Introduction: Potent, selective, and orally available small molecule inhibitors of the p53 regulator MDM2 represent a promising class of therapeutics. When applied to p53WT tumors, MDM2 inhibitors disrupt the MDM2-p53 interaction, thereby activating the p53 pathway and inducing cell cycle arrest and apoptosis. The aim of this study was to evaluate two clinically available PET tracers for their ability to monitor early response to treatment with a novel MDM2 inhibitor. Methods: 2’-Deoxy-2’-18F-fluoro-D-glucose (FDG) is used to measure glucose transport and hexokinase activity, and its use in cancer detection and response is based on the preferential glycolytic metabolism of tumor cells. 3′-Deoxy-3′-18F-fluoro-L-thymidine (FLT), an analog of thymidine, can measure nucleoside transport and thymidine kinase activity, which is upregulated in the S-phase of mitosis and can serve as a surrogate for cellular proliferation. Two p53WT in vivo tumor models were evaluated: (1) MDM2-amplified SJSA-1 human osteosarcoma xenografts which exhibit tumor regression upon MDM2 inhibitor treatment, and (2) MDM2-WT HCT116 human colon carcinoma xenografts which exhibit tumor stasis upon treatment. FDG and FLT PET scans (separate cohorts) were acquired (1) 24 hr before treatment with MDM2 inhibitor or vehicle (i.e., baseline), (2) after 6 hr and 72 hr of treatment, and (3) 72 hr after cessation of treatment (i.e., wash-out). Results: MDM2 inhibitor treatment resulted in comparable decreases in FDG and FLT tumor standardized uptake values (SUV) in SJSA-1 tumor xenografts at 6 hr post-treatment (-23% and -9% relative to baseline, respectively; P<0.01) that persisted throughout the 72 hr treatment (-52% and -34%, respectively; P<0.001) and 72 h wash-out period (-54%; P<0.0005 and -28%, respectively). In HCT116 xenografts, MDM2 inhibitor treatment significantly reduced FLT tumor SUV at 6 hr post-treatment (-32% relative to baseline, P<0.005) and persisted throughout the 72 hr treatment (-70%; P<0.001), and rebounded sharply by the end of the wash-out period in HCT116 xenografts (-32%; P<0.005). FDG tumor SUV was not significantly reduced at any timepoint in HCT116 xenografts. Conclusions: FDG and FLT PET imaging were roughly equivalent in ability to monitor efficacy of MDM2 inhibitor treatment in a SJSA-1 tumor regression model; however only FLT PET imaging revealed a treatment effect in the HCT116 tumor stasis model. FLT PET imaging was successfully used as a noninvasive tool to provide quantitative measurement of tumor response to treatment with a novel MDM2 inhibitor. These results suggest that FLT PET imaging could have utility as an early clinical pharmacodynamic biomarker. Citation Format: Tim Kazules, Becky Bryant, Matt Silva, Jude Canon, Charles Glaus. 18F-FLT PET imaging for noninvasive, early assessment of tumor response to MDM2-p53 disruption using a novel MDM2 inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2658. doi:10.1158/1538-7445.AM2013-2658

Introduction: Dysregulation of the hepatocyte growth factor (HGF)/MET pathway has been implicated in various cancers. Rilotumumab is an investigational, fully human monoclonal antibody that binds and neutralizes HGF. The purpose of this study was to evaluate the efficacy of rilotumumab in a U-87 MG mouse xenograft tumor model using F-18-FDG and F-18-FLT PET.Methods: U-87 MG tumor-bearing nude mice received rilotumumab or control IgG2. In the dose response study, increasing doses of rilotumumab (10, 30, 100, 300, or 500 mu g) were administered, and mice were evaluated with F-18-FDG PET at baseline and 7 days post-treatment. In the time course study, 300 mu g of rilotumumab twice per week was used for the treatment, and mice were evaluated over 7 days using F-18-FDG and F-18-FLT PET.Results: In the dose response study, rilotumumab at doses of 300 and 500 mu g was similarly effective against tumor growth. Treatment with 300 and 500 mu g rilotumumab inhibited F-18-FDG accumulation with significant decreases of 37% and 40% in the percent injected dose per gram of tissue (%ID/g), respectively. In the time course study, treatment with 300 mu g rilotumumab inhibited F-18-FDG and F-18-FLT accumulation with a maximum %ID/g of 41% and 64%, respectively. No apparent differences between the use of either tracer to evaluate rilotumumab efficacy were observed.Conclusions: Rilotumumab inhibited F-18-FDG and F-18-FLT accumulation as early as 2 and 4 days after treatment, respectively, in a mouse tumor model. Further studies to evaluate F-18-FDG PET imaging as an early tumor response marker for rilotumumab are warranted. Rilotumumab is currently being tested in patients with MET-positive, advanced gastric and gastroesophageal cancer. (C) 2013 Elsevier Inc. All rights reserved.

Positron emission tomography (PET) imaging with the glucose analog 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F] FDG) has demonstrated clinical utility for the monitoring of brain glucose metabolism alteration in progressive neurodegenerative diseases. We examined dynamic [(18)F]FDG PET imaging and kinetic modeling of atlas-based regions to evaluate regional changes in the cerebral metabolic rate of glucose in the widely-used 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. Following a bolus injection of 18.5 ± 1 MBq [(18)F]FDG and a 60-minute PET scan, image-derived input functions from the vena cava and left ventricle were used with three models, including Patlak graphical analysis, to estimate the influx constant and the metabolic rate in ten brain regions. We observed statistically significant changes in [(18)F]FDG uptake ipsilateral to the 6-OHDA injection in the basal ganglia, olfactory bulb, and amygdala regions; and these changes are of biological relevance to the disease. These experiments provide further validation for the use of [(18)F]FDG PET imaging in this model for drug discovery and development.

Gold nanocages represent a novel class of nanostructures, well-suited for biomedical applications. They can be readily prepared via the galvanic replacement reaction between silver nanocubes and chloroauric acid. Their optical resonance peaks can be easily and precisely tuned to the near-infrared region from 650-900 nm, the transparent window for blood and soft tissue. Furthermore, their surface can be conveniently conjugated with various ligands for targeting cancer. In this feature article, we highlight recent advances in the large-scale synthesis of gold nanocages and their applications in cancer diagnosis and treatment. Specifically, we have scaled up the production of gold nanocages for in vivo studies and evaluated their tumor targeting capabilities. We have also demonstrated their use as contrast agents for photoacoustic tumor imaging and the mapping of sentinel lymph node, as photothermal transducers for cancer treatment, and as smart carriers for controlled release with a near-infrared laser.

A novel nanoparticle-based dual-modality positron emission tomograph/magnetic resonance imaging (PET/MRI) contrast agent was developed. The probe consisted of a superparamagnetic iron oxide (SPIO) core coated with PEGylated phospholipids. The chelator 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to PEG termini to allow labeling with positron-emitting Cu-64. Radio labeling with Cu-64 at high yield and high purity was readily achieved. The (Cu-64-SPIO probes produced strong MR and PET signals and were stable in mouse serum for 24 h at 37 degrees C. Biodistribution and in vivo PET/CT imaging studies of the probes showed a circulation half-life of 143 min and high initial blood retention with moderate liver uptake, making them an attractive contrast agent for disease studies.