Found 14 Records SESSION TITLE: Innovation of the Year, Coffee Break & Visit ExhibitsCONTROL ID: 2805290TITLE: High-resolution, tomographic imaging of adeno-associated virus (AAV) transgene expression in live animalsusing the sodium iodide symporter (NIS) reporter genePRESENTER: Lukkana SuksanpaisanAUTHORS (FIRST NAME, LAST NAME): Jonathan K. Mitchell1, Jessica H. Newman1, Toshie Sakuma1, Linh Pham1

, Stephen Russell1, Kah-Whye Peng1, Lukkana Suksanpaisan1

INSTITUTIONS (ALL): 1. Imanis Life Sciences, LLC, Rochester, MN, United States.ABSTRACT BODY: Abstract Body: Although recently-developed molecular tools, such as CRISPR/Cas9, have revolutionized the field ofgene therapy, significant challenges remain for directing gene therapy vectors to diseased organs or tissues with highspecificity in vivo. Adeno-associated virus (AAV) vectors engineered for organ-specific tropism or transgeneexpression have the potential to yield safe and efficacious routes for gene therapy delivery. However, traditionalmethodologies for tracking AAV biodistribution, such as quantitative PCR (qPCR) and immunohistochemistry (IHC),are invasive, costly, and time-consuming, and these drawbacks pose significant limitations towards developing andevaluating novel AAV vectors. Reporter genes, particularly firefly luciferase, have been used as a non-invasivealternative for monitoring AAV transgene expression, but such reporters are typically low-resolution, immunogenic,and suitable for use only in small animal models.The sodium iodide symporter (NIS) is a self-protein expressed primarily in the mammalian thyroid and stomach.Beyond its physiological role in trapping iodide within cells, NIS can function as a reporter gene for high-resolution,tomographic SPECT or PET imaging via its ability to drive cellular uptake of radiotracers. Unlike luciferase, NISreporter gene imaging can be used in small and large animal models, as well as humans, thus offering the ability touse a single reporter gene to guide studies from pre-clinical to clinical phases. Given these advantages, we sought toinvestigate the utility of NIS as a reporter gene for non-invasive tracking of AAV transgene expression in vivo. To thisend, we generated an AAV9 vector expressing NIS (AAV9-NIS) and delivered this vector intravenously into nudemice. Non-invasive imaging with the PET radiotracer 18F-tetrafluorobate (18F-TFB) revealed high-level NIS reportergene expression within the heart and liver, consistent with the established tropisms of AAV9. NIS transgeneexpression was also observed within the brown fat and throughout the skeletal muscle of AAV9-NIS mice. In contrast,background signal in control mice was restricted to sites of endogenous NIS expression (e.g. thyroid and stomach).IHC analyses of NIS expression in organs harvested from AAV9-NIS mice confirmed NIS expression in the heart,liver, and muscle, consistent with our PET imaging data. Combined, these data establish NIS reporter gene imagingas a powerful, non-invasive alternative to traditional AAV biodistribution assays.(No Image Selected)

SESSION TITLE: Late Breaking Session 05: Basic Bio - New Probe & Systems/Engineered BiologyCONTROL ID: 2782551TITLE: Nuclear imaging of cancer or immune cell derived extracellular vesicles after radioiodine labelingPRESENTER: Chae Moon HongAUTHORS (FIRST NAME, LAST NAME): Chae Moon Hong1, Prakash Gangadaran1, JIMIN OH1, Ramya LakshmiRajendran1, Liya Zhu1, Beak Se Hwan1, SENTHILKUMAR KALIMUTHU1, Ho Won Lee1, Sang Bong Lee1, YongHyun Jeon1, Shin Young Jeong1, Sang-Woo Lee1, Jaetae Lee1, Byeong-Cheol Ahn1

INSTITUTIONS (ALL): 1. Nuclear Medicine, Kyungpook Nation University, school of medicine and hospital, Daegu, Korea (the Republic of).ABSTRACT BODY: Abstract Body: ObjectivesBiodistribution and role of extracellular vesicles (EVs) are still largely unknown, and reliable tracking methods for EVsare needed. In this study, nuclear imaging using radioiodine were developed and applied for tracking EVs derivedfrom anaplastic thyroid cancer (CAL-62) and dendritic cells.MethodsEVs were obtained from supernatant of CAL-62 cell media and dendritic cells using sequential ultracentrifuges.Sulfosuccinimidyl-3-(4-hydroxypheynyl) propionate were labeled to membrane of CAL-62 and dendritic cell derivedEVs, then the EVs were labeled with radioiodine (I-123, I-124 and I-131) pre-coated iodination tubes (RI-EVs). In-vivogamma camera and PET/CT images were obtained after intravenous injection of the RI-EVs.ResultsWe successfully labeled EVs with radioiodine and radiochemical purity of the RI-EV was 98.9±2.2%. Results ofnanoparticle tracking analysis and transmission electron microscopy showed that there was no significant difference inEVs before and after the radiolabeling. After intravenous injection of RI-EVs to mice, gamma camera and PET/CTimaging well visualized real-time biodistribution of the RI-EVs.ConclusionsWe established a nuclear imaging system of EVs derived from thyroid cancer and dendritic cells using radiolabeling ofthe EVs, and this system might be helpful for in vivo tracking of EVs.(No Image Selected)

SESSION TITLE: Late Breaking Session 05: Basic Bio - New Probe & Systems/Engineered BiologyCONTROL ID: 2804232TITLE: Triple reporter redux: A fluorescent, bioluminescent, and PET reporter construct for preclinical imaging.PRESENTER: Mark SellmyerAUTHORS (FIRST NAME, LAST NAME): Mark A. Sellmyer1, Sarah A. Richman2, 1, Katheryn Lohith1, Catherine Hou1, David Mankoff1, Robert H. Mach1, Micheal C. Milone1, Michael D. Farwell1

INSTITUTIONS (ALL): 1. University of Pennsylvania, Philadelphia, PA, United States. 2. Children's Hospital of Philadelphia, Philadelphia, PA, United States.ABSTRACT BODY: Abstract Body: Preclinical imaging is often used for validating a particular hypothesis in live animals. Traditionalimaging modalities such as bioluminescent imaging, intravital microscopy, and small animal PET have different spatialresolution and procedural parameters, and can often be used in a coordinated fashion to provide additional layers ofgranularity to in vivo data.1 A recently developed a small molecule PET reporter gene and probe pair, based onbacterial dhfr and C-11 and F-18 radiolabeled derivatives of the small molecule trimethoprim ([11C]TMP and[18F]FPTMP), has attractive features in combination including the probe biodistribution, metabolism and excretion aswell as its sensitivity for detecting relatively small numbers of transgenic cells.2,3 Here, we describe the generation ofa triple imaging genetic reporter construct (DYR) based on a dhfr-YFP fusion protein with 2A ribosomal skip sitefollowed by Renilla luciferase. Lentiviral transduction of ex vivo expanded primary T cells was performed and cellswere selected for YFP expression using FACS. We then show over 50-fold bioluminescent signal induction in DYR Tcells compared to control T cells. Additionally, we show over 10-fold in vitro increased uptake after incubation with[18F]FPTMP for 2h in DYR T cells as compared to control T cells, normalizing to cell number as well as proteinconcentration. In vivo testing of the construct in small animal models is on-going. In summary, we believe this triplereporter construct with a recently developed PET reporter gene and probe pair may be useful for in vivo imaging of awide variety of cells and across many different biologic disciplines. 1. Kim YJ, Dubey P, Ray P, Gambhir SS, Witte ON. Multimodality imaging of lymphocytic migration using lentiviral-based transduction of a tri-fusion reporter gene. Mol Imaging Biol. 2004;6(5):331-340.2. Sellmyer MA, Lee I, Hou C, et al. Quantitative PET Reporter Gene Imaging with [11C]Trimethoprim. Mol Ther.2017;25(1):120-126.3. Mark A. Sellmyer IL, Catherine Hou , Chi-Chang Weng , Shihong Li , Brian P. Lieberman , Chenbo Zeng , David A.Mankoff , Robert H. Mach. Bacterial Infection Imaging with [18F]Fluoropropyl-trimethoprim. Proc Natl Acad Sci U S A.2017 (In Press).(No Image Selected)

SESSION TITLE: Late Breaking Session 05: Basic Bio - New Probe & Systems/Engineered BiologyCONTROL ID: 2805267TITLE: Engineering De Novo Fluorescent Bili-Proteins via Computationally Driven DesignPRESENTER: Ivan KuznetsovAUTHORS (FIRST NAME, LAST NAME): Ivan A. Kuznetsov1, Michael S. Magaraci1, Molly M. Sheehan1, Brian Y.Chow1

INSTITUTIONS (ALL): 1. Bioengineering, University of Pennsylvania, Philadelphia, PA, United States.ABSTRACT BODY: Abstract Body: Recently, we reported the creation of genetically encoded molecular imaging agents using de novofour-helix bundle proteins that are mammalian cell-expressible.1 This artificial protein platform offers a fundamentallynew way of designing optogenetic tools and sensors from the “bottom-up,” i.e. from first principles. However, giventhat these proteins are de novo, they lack the evolutionary optimization seen in natural structures and knownhomology models to guide engineering efforts. Increasingly powerful computational protein design tools are well-posed to bridge this structure-function gap.2 Here, we report the creation of two de novo infrared fluorescent proteins(dFPs)1: the first (dFP-2.0) by computational redesign of a crystallized heme-binding de novo four-helix bundle toinstead incorporate a biliverdin chromophore, and the second (dFP-mini) by iterative ab initio computational andempirical design from our previously reported biliverdin-binding dFP1.The Rosetta design suite was used for computational design to relax a biliverdin-binding pocket into an initial ab initio3

model (dFP-mini) or into a crystallized heme-bound four-helix bundle (dFP-2.0). The design process proceeded viatwo main steps. First, the Rosetta matcher and ligand docking algorithms4,5 were used to find a structure that couldsatisfy several geometric constraints necessary for fluorescent holoprotein formation, namely covalent attachment ofbiliverdin to a targeted cysteine residue, minimal solvent exposure of the chromophore within the hydrophobic core,and significant hydrogen bond interaction between chromophore and protein scaffold. Next, Rosetta’s enzyme designprotocol6 was used to further stabilize the biliverdin chromophore without altering the previously imposed geometricconstraints or permitting a higher affinity competitive binding by heme. Over a million possible structures were virtuallyscreened and several leads were selected for experimental validation.The resulting far-red fluorescent proteins (λex ~645nm, λem ~660nm) were validated by spectroscopy of purifiedproteins and expression in HEK cells, and they possess similar properties in mammalian cells as those derived fromnatural bacteriophytochromes despite lacking sequence and structural homology to fluorescent bili-proteins derivedfrom natural scaffolds.7,8 Due to their compactness, as small as 9kD for dFP-mini or ~one-third the size of GFP, dFPtechnologies show great promise for use in deep tissue imaging and as AAV-mediated gene therapy tags. 1. Sheehan, M. M. et. al. WMIC, ID: 2491848 (2016).2. Huang, P. S. et al. Nature 537, 320-327 (2016).3. Raman, S. et. al. Proteins 77, 89-99 (2009).4. Zanghellini, A. et al. Protein Science 15, 2785–2794 (2015).5. Meiller, J. et al. Proteins 65, 538-548 (2006).6. Richter, F. et. al. PLoS ONE 6, e19230 (2011).7. Shu, X. et al. Science 324, 804–807 (2009).8. Filonov, G. S. et al. Nature Biotechnology 29, 759–763 (2011).(No Image Selected)

SESSION TITLE: Poster Session 01CONTROL ID: 2727033TITLE: In vitro assessment of a novel PSMA-targeted nanobubble for prostate cancer ultrasound imagingPRESENTER: Afsana AkhterAUTHORS (FIRST NAME, LAST NAME): Afsana Akhter3, Jacob Lilly3, Christopher Hernandez3, GopolakrishnanRamamurthy2, Hansheng xia3, Xinning Wang1, Agata A. Exner4, James Basilion4

INSTITUTIONS (ALL): 1. Case Western Reserve University, Cleveland, OH, United States. 2. Radiology, Case Western Reserve University, Cleveland, OH, United States. 3. Department of Radiology, Case Western Reserve University, Cleveland Heights , OH, United States. 4. Radiology and Biomedical Engineering, Case Western Reserve University, Cleveland, OH, United States.ABSTRACT BODY: Abstract Body: Abstract: Prostate cancer (PCa) biopsies are commonly performed using ultrasound (US) guidance.Because soft tissue contrast in typical US images is poor, tumors can be difficult to discern. There is thus an urgentneed to develop a more effective tool to delineate cancer accurately within the prostate gland for PCa managementand biopsy guidance. To meet this need we have developed a sub-micron lipid and surfactant-stabilized US contrastagent (nanobubble or NB) targeted to the prostate specific membrane antigen (PSMA) via a highly selective ligand(PSMA-1)2. In comparison to microbubbles (MB), which remain in the vasculature, this targeted NB (<300 nm) 3,4 willbe able to extravasate into the tumor parenchyma and directly bind to the cancer cells. This will result in higheraccumulation of contrast agent at the tumor leading to better resolution and detection of PCa. Here we report on invitro characterization of the PSMA-1-NB construct. Lipid conjugation of PSMA-1 was performed through the γ-NH2 oflysine. PSMA-1 was dissolved in anhydrous DMSO, to which 2.5-fold excess amount of Maleimide-PEG(2k)-DSPEwas added. To formulate NBs lipids DPPC, DPPE, DPPA and DSPE-PEG-PSMA-1 were dissolved in chloroform at a4:1:1:1 ratio, dried and hydrated in PBS with Pluronic L10 solution. NBs were fluorescently tagged by adding DiI intothe lipid film. To determine cell binding, PSMA-expressing cells (PC3pip) and cells that do not express PSMA (PC3flu)were seeded (1x106 cells) onto 35mm dishes containing a cover slip 24 hrs prior to the experiment. Incubation of NBswith cells in culture for 60 min showed that targeted NBs accumulated significantly higher in PC3pip cells (Fig-1). Datawere collected using a fluorescent microscope and images processed to quantify fluorescence signal in cells. An invitro competition binding assay was also carried out. Here, 5X 105 cells were incubated with different conc. of PSMA-1-NB in presence of 12 nmol/L N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-[3H]-methyl-L-cysteine for 1hr. After incubation cells were washed 3 times with PBS and finally 4 ml of EcoLumecocktail was added and radioactivity was counted. Results show that the IC50 of PSMA-1- NB is lower (9.2 nM) thanthe ligand PSMA-1 (18.6 nM) (Fig 2) in PSMA positive LNCaP cells. These preliminary in vitro data suggest that thesePSMA targeted nanobubbles may become useful as targeted US contrast agents for PSMA positive PCamanagement in the future. References1. Hankey et al. Journal of the National Cancer Institute 1999; 91(12): 1017-24.2. Wang et al. Mol Cancer Ther 2014; 13(11): 2595-606.3. Perera RH et al. Nanomedicine 2017 Jan;13(1):59-674. Wu H et al, Ultrasound Med Biol 2013 Nov;39(11):2137-46

IMAGE CAPTION:

SESSION TITLE: Poster Session 01CONTROL ID: 2727100TITLE: Longitudinal imaging of cationic lipid nanoparticle based vaccine formulation reveal enhanced antigenretention at the injection sitePRESENTER: Shu-An LinAUTHORS (FIRST NAME, LAST NAME): Shu-An Lin2, Gokul Swaminathan3, 4, Marian Gindy1, Andrew J. Bett3,Manishkumar Patel2

INSTITUTIONS (ALL): 1. Pharmaceutical Sciences, Merck & Co., Inc., West Point, PA, United States. 2. Translational Biomarkers, Merck & Co., Inc., West Point, PA, United States. 3. Infectious Diseases and Vaccine Research, Merck & Co., Inc., West Point, PA, United States. 4. Immuno-Biology, Merck & Co., Inc., Cambridge, MA, United States.ABSTRACT BODY: Abstract Body: Emerging evidence suggests that bioengineered nanoparticles can be used as immunomodulatoryagents. We have identified unique ionizable cationic lipid nanoparticle (LNPs) formulations that possess the ability toenhance B-cell and T-cell responses to sub-unit vaccine antigens[1]. The level of vaccine responses achieved with ourLNPs is far superior to those achieved with known vaccine immune-modulators in preclinical animal models.Furthermore, the effect was independent of any known toll-like receptor agonists formulated with the LNPs. It isimperative to understand the mechanism of action (MOA) of these LNPs for the effective use of this novel immune-modulator in the development of immunotherapeutic vaccines against various infectious diseases and cancer. Toinvestigate the MOA, we utilized a fluorescently tagged model antigen, Ovalbumin (OVA) in a murine model ofintramuscular vaccine administration. Through tracking the biodistribution of OVA with or without LNPs, we sought todetermine whether LNPs enhance retention time of an antigen at the site of injection.Groups of female SKH1-E mice were injected intramuscularly with Alexa Fluor 700 labeled ovalbumin (OVA-AF700)(10ug) with or without Alexa Fluor 790 labeled non-specific siRNA encapsulated within Merck proprietary LNPs (LNP-siR-AF790). Animals were imaged at 0, 24, 48, 72 and 168 h post-injection using an IVIS Spectrum. The OVA-AF700signal was statistically significantly higher at the injection site when co-injecting with LNPs at 24 h time point and stillvisible up to day 3 post-injection (Figure 1), which indicates that LNP enhances antigen (OVA) retention at theinjection site. In addition, LNP-siR-AF790 remained at the injection throughout the study independent of the presenceof OVA-AF700 although some LNP-siR-AF790 was detected in liver. In separate groups of animals, we furtherevaluated the inflammatory cytokines and chemokines in the peripheral serum and the injection site (muscle). LNPsinduced a localized chemokine response at the site of injection rather than systemic peripheral inflammation, whichsupports the notion that LNPs might be inducing a chemotactic gradient at the injection site and recruit specificimmune cells that aid in boosting B-cell and T-cell responses to the antigen of interest. Immuno-profiling of innateimmune cells in the draining lymph nodes post vaccination revealed that LNPs effectively delivered the co-administered antigen to the lymph-node-resident dendritic cells (DCs). In summary, by tracking the biodistribution ofLNP-antigen vaccine formulation, we uncovered that enhanced retention of antigen at the injection side and antigendelivery to DCs might be critical factors for its immune-modulatory properties. Such detailed knowledge of LNP’smode of action will significantly influence the future design of LNPs for vaccines and immune oncology applications. References:[1] Swaminathan G, Thoryk EA, Cox KS, Meschino S, Dubey SA, Vora KA, et al. A novel lipid nanoparticle adjuvantsignificantly enhances B cell and T cell responses to sub-unit vaccine antigens. Vaccine Jan 02;34(1):110-9.

Figure 1 In vivo quantification of OVA-AF700 signal at the injection site

IMAGE CAPTION: Figure 1 In vivo quantification of OVA-AF700 signal at the injection site

SESSION TITLE: Poster Session 01CONTROL ID: 2731174TITLE: Evaluation of the potential of minoxidil coated lysozyme shelled microbubbles combine with ultrasound topromote mouse hair follicle growthPRESENTER: Yu-Jhen HuangAUTHORS (FIRST NAME, LAST NAME): Yu-Jhen Huang1

INSTITUTIONS (ALL): 1. Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Taipei,Taiwan.ABSTRACT BODY: Abstract Body: In our previous study, a new type of minoxidil (Mx) coated albumin shelled microbubbles (MBs) andcombined them with sonication by ultrasound energy in the water phase was created to enhance hair growth.Lysozyme (Lyz) is an antimicrobial peptide, and recently, it was also used to stimulate hair follicle growth. In thisstudy, a new minoxidil coated lysozyme shelled MBs (LyzMBs) was produced to inhibit bacterium or the allergycaused in oil scalp. Moreover, the potential of minoxidil coated lysozyme shelled MBs combine with US either toenhance hair growth or to promote the number of hair follicle was investigated.

IMAGE CAPTION:

SESSION TITLE: Poster Session 01CONTROL ID: 2735201TITLE: Towards molecular imaging of synthetic biological circuits in mammalian cellsPRESENTER: jangsun hwangAUTHORS (FIRST NAME, LAST NAME): jangsun hwang3, 1, Jonghoon Choi1, Galit Pelled2, Assaf A. Gilad3

INSTITUTIONS (ALL): 1. SCHOOL OF INTEGRATIVE ENGINEERING, Chung-Ang University, Seoul, Seoul, Korea (the Republic of). 2. Johns Hopkins/Kennedy Krieger Institute, Baltimore, MD, United States. 3. Johns Hopkins School of Medicine, Baltimore, MD, United States.ABSTRACT BODY: Abstract Body: Background and significance: Developing synthetic biological devices that can non-invasively controlcell fate and function in vivo can revolutionize the field of regenerative medicine 1,2. The ability to visualize the outputof such biological devices using molecular imaging tools will allow fine-tuning of biological devices with the outmostprecision. To address this unmet need we have designed a biological circuit consisting of three bioparts (1) as aninput an “on”/off switch”, (2) a “biological amplifier” and (3) reporter gene for readout. We have recently cloned a genethat encodes to a putative membrane associated protein that responds to electromagnetic fields (EMF).Electromagnetic perceptive gene (EPG) was isolated from the Kryptopterus bicirrhis (glass catfish), which increasesintracellular calcium ions by stimulation of EMF. We seek to develop a system for a remote regulation of (reporter)gene expression by EMF from generated streptavidin-coated ferromagnetic nanoparticles (FMNPs). Here wedemonstrate that the EPG can be used as a molecular switch as a first step for constructing biological circuit inmammalian cells. Methods: HEK293T cells were transfected with a plasmid pCDNA3.1CMV::EPG-IRES-GFP. Cellular response wasmeasured as increase in intracellular calcium using the Ca2+ indicator (fura-2/AM). For imaging of cells,340nm/380nm ratio was calculated to measure intracellular calcium change. Biotin was conjugated to amine groups ofcell surface proteins of engineered HEK293TGFP-EPG and control HEK293TGFP cells as described before3. FMNPs[Spherotech, Inc. USA] or as control superparamagnetic nanoparticles (SMNPs [Spherotech, Inc. USA]) conjugated tostreptavidin were added to the cells (Figure 1 A).Results: Figure 1 B,C demonstrates that only biotinylated cells that expressed GFP-EPG (HEK293TGFP-EPG) thatwere stimulated with FMNPs showed a significance increase in intracellular calcium levels while control cells –eitherbiotinylated HEK293TGFP-EPG stimulated with SMNPs or biotinylated HEK293TGFP stimulated with FMNPs-showed no change in calcium levels. Moreover, 39± 14% of the HEK293TGFP-EPG showed significant increase incellular calcium (5 times greater than the standard deviation) in response to FMNPs stimulation (Figure 1 D). Incontrast, none of the controls showed significant change in calcium. These results indicate that the EPG activated withFMNP can be used as a biological device switch that can be visualized in vivo.Conclusion: Our finding is the first step toward the development of synthetic biological circuits in mammalian cells. Inthe future, such devices can be combined with calcium sensitive promoters4,5 driving genetically encoded reportersfor a variety of imaging modalities for noninvasive imaging in live animals.2 1. Ruder, W. C., Lu, T. & Collins, J. J. Science 333, 1248-1252, (2011); 2. Gilad, A. A. & Shapiro, M. G. Mol ImagingBiol, (2017); 3. Hwang, J., Seo, Y., Jo, Y., Son, J. & Choi, J. Sci Rep 6, 34778, (2016); 4. Jouroukhin, Y., Nonyane, B.A., Gilad, A. A. & Pelled, G. J Mol Neurosci, (2014); 5. Merlet, E. et al. Gene Ther 20, 248-254, (2013)(No Image Selected)

SESSION TITLE: Poster Session 01CONTROL ID: 2735239TITLE: Re-engineering of Self-assembly Interfaces with Metal Ion Coordination In A Protein Nanocage for ExtendedBiofunctions PRESENTER: Zhantong WangAUTHORS (FIRST NAME, LAST NAME): Zhantong Wang2, Zhe Wang2, Orit Jacobson2, Fuwu Zhang2, Bryant C.Yung2, Gang Niu2, Gang Liu3, Xiaoyuan Chen1

INSTITUTIONS (ALL): 1. NIBIB/CC/NIH, Bethesda, MD, United States. 2. NIBIB, National Institutes of Health, Bethesda, MD, United States. 3. State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular Imaging andTranslational Medicine, Xiamen University, Xiamen, Fujian, China.ABSTRACT BODY: Abstract Body: The bottom-up self-assembly of protein subunits into supramolecular architectures is ubiquitouslyexploited to construct natural protein macromolecules as well as artificial designer proteins. The accumulativeknowledge of both protein self-assembly mechanisms and coherent interactions of subunits enables us to utilize thisstrategy to advance medicine and material science. A defined re-engineering strategy, thus, is required for a givenprotein to expand its functions in practice. The chemical or biological re-engineering methods are available to renderproteins diverse functions, they are, unfortunately, undertaking the risk of compromising protein integrity and stabilityafter extensive modifications. In this study, we introduced a new protein engineering method, metal ion assistedinterface re-engineering, to serve as a robust, straightforward and universal strategy to extend biofunctions of a self-assembly protein. This re-engineering strategy was applied to a widely used natural protein, ferritin, as a model self-assembly protein to coordinate with copper ions in the mutagenic artificial metal binding domain in the proteinstructure. Structured directed rational protein mutagenesis was carried out at the C2 interface amino acid residues ofthe ferritin subunit for metal ion coordination site optimization. Copper binding at the artificial binding pocket is specificover other divalent ions available in physiological fluids, and the structurally embedded copper ion in turnstrengthened the overall protein integrity and stability. In presence of isotopic copper-64, the interface re-engineeredferritin worked as a chelator free molecular nanoprobe with surprisingly high specific radioactivities to enableconsistent high-resolution PET imaging of tumor in living animals. We envision that the metal ion assisted interface re-engineering could provide a new approach to re-construct self-assembly proteins with unachieved properties andextended functions in broad biomedical applications.

IMAGE CAPTION:

SESSION TITLE: Poster Session 01CONTROL ID: 2735736TITLE: Non-Covalent Polymer Shell Encapsulation of Immunomodulatory Lipids in Ultrasmall Paramagnetic IronOxide NanoparticlesPRESENTER: Evan StaterAUTHORS (FIRST NAME, LAST NAME): Evan Stater1, Edwin C. Pratt1, Jan Grimm2

INSTITUTIONS (ALL): 1. Pharmacology, Weill Cornell Graduate School of Medical Sciences, New York, NY, United States. 2. Molecular Pharmacology and Chemistry, memorial Sloan kettering Cancer Center, New York, NY, United States.ABSTRACT BODY: Abstract Body: Extremely hydrophobic lipid drugs often exhibit adverse pharmacokinetic properties, which areproblematic to therapeutic applications. Complexation of therapeutic lipids within a carrier nanophore particle may bea useful strategy for improving pharmacologic parameters. We described previously that small organic moleculepayload compounds can be loaded into the polymer shell of ferumoxytol (Feraheme, FH), an FDA-approved ultrasmallparamagnetic iron oxide nanoparticle (Kaittanis et al. Nat Comms. 2014). Here, we now follow up on this work anddemonstrate that lipidic Toll-like receptor ligands (LTLRL) can also be loaded stably into FH nanoparticles. Magnetically-separated FH fractions exhibit differential drug uptake capacity for LTLRL, with a correlation observedbetween magnetic column particle affinity and particle drug capacity. The physical differences in particle structurewhich influence this differential uptake are subject of further study. We propose that the nanoparticle subpopulationwhich is most strongly retained in the magnetic separation column exhibits a physical characteristic or combination ofcharacteristics which simultaneously improve ferromagnetic attraction to the column bed, and facilitate drugencapsulation in the polymer coating. The Toll-like receptor agonist activity of immunostimulatory LTLRLs are retained in complex with FH. FH-LTLRLnanoparticles exhibited potent stimulatory activity on cultured murine leukocytes, as shown by assays of leukocyteactivation and immunomodulatory cytokine secretion. This represents a promising and translationally-relevantapproach for the use of FH in immunomodulatory theranostic applications, such as cancer immunotherapy. In vivostudies to evaluate LTLRL delivery and therapeutic immunomodulation in tumors are planned.(No Image Selected)

SESSION TITLE: Poster Session 03CONTROL ID: 2805129TITLE: Genetically controlled subcellular localization of modified ferritin nanocages enables multimodal non-invasiveimaging and actuationPRESENTER: Christoph MassnerAUTHORS (FIRST NAME, LAST NAME): Christoph Massner1, 2, 3, Felix Sigmund1, 2, Susanne Pettinger1, 2,Markus Seeger1, 4, Carolin Hartmann5, Natalia P. Ivleva5, Hannes Rolbieski1, 2, Vasilis Ntziachristos1, 4, UlfWiedwald6, Michael Winklhofer7, Gil G. Westmeyer1, 2, 3

INSTITUTIONS (ALL): 1. Institute of Biological and Medical Imaging, Helmholtz Zentrum München, Neuherberg, Germany. 2. Institute of Developmental Genetics, Helmholtz Zentrum München, Neuherberg, Germany. 3. Department of Nuclear Medicine, Technical University of Munich, Munich, Germany. 4. Chair for Biological Imaging, Technische Universität München, Munich, Germany. 5. Chair of Analytical Chemistry & Institute of Hydrochemistry, Technical University of Munich, Munich, Germany. 6. Faculty of Physics and Center for Nanointegration (CENIDE), University of Duisburg-Essen, Duisburg, Germany. 7. Institute for Biology and Environmental Sciences IBU, University of Oldenburg, Oldenburg, Germany.ABSTRACT BODY: Abstract Body: Genetically controlled interfaces for non-invasive imaging and manipulation techniques are importantand highly demanded tools to understand the correlative and causal relationships in cellular networks. Herein wedemonstrate the utility of ferritin importers in combination with unmodified eukaryotic ferritin and synthesizedmagnetoferritin for several non-invasive imaging and manipulation techniques. Lysosomal trafficking of ferritin variantsaltered magnetic properties of the interfaces and enabled their multimodal and multiscale use for imaging andactuation techniques. Ferritin heavy chain receptors TfR1 and Tim-2, as well as the light chain receptor Scara5, werecompared regarding their performance to take up native ferritin (eFTH+L) or magnetoferritin (hFTH-Mag). To assessthe performance of the different receptors in combination with these two ferritin variants, we used magnetic resonanceimaging (MRI) and magnetic sorting on superparamagnetic columns. We demonstrate that only cells overexpressingTim-2 show efficient uptake of both eFTH+L and hFTH-Mag compared to cells overexpressing TfR1 or Scara5.Therefore, Tim-2 overexpressing cells were used for further experiments demonstrating a range of imaging andactuation techniques based on lysosomal encapsulation of ferritin. The controlled uptake of highly magnetized hFTH-Mag enabled magnetic manipulation of single cells through an electromagnetic needle and could be used for magnetichyperthermia induced cell death. We furthermore evaluated the semi-genetic system via viral transduction in murinebrains. These findings demonstrate a broad range of possible applications for the bioengineered interface enablingboth non-invasive multimodal imaging and actuation. The biomagnetic interface can be tailored either genetically orbiosynthetically by using different receptors or exchanging the ferritin core and is a versatile tool to interrogate cellularnetworks remotely.(No Image Selected)

SESSION TITLE: Scientific Session 15: Basic Biology - Imaging BiochemistryCONTROL ID: 2724633TITLE: A novel protein based near infrared molecular imaging probe based on low fouling and biodegradable PASKEparticlesPRESENTER: Thomas BonnardAUTHORS (FIRST NAME, LAST NAME): Thomas Bonnard3, Jasmine A. Putri3, Jiwei Cui1, 2, Yi Ju1, KarlinePascaud3, Hannah Pearce3, Shweta Jagdale3, Frank Caruso1, Christoph Hagemeyer3

INSTITUTIONS (ALL): 1. ARC Centre of Excellence in Convergent Bio-Nano Science and Technology and the Department of Chemical andBiomolecular Engineering, The University of Melbourne, Melbourne, VIC, Australia. 2. Key Laboratory of Colloid and Interface Chemistry of Ministry of Education and the School of Chemistry andChemical Engineering, Shandong University, Jinan, Shandong, China. 3. Australian Centre for Blood Diseases, NanoBiotechnology Laboratory, Monash University, Melbourne, VIC,Australia.ABSTRACT BODY: Abstract Body: Nanomedicine holds great promises for vascular disease diagnostics and specific therapies butmaterials based on non-natural polymers might accumulate in the human body and cause adverse effects afterprolonged use. Thus, new biocompatible nanoparticle formulations are required to enable clinical translation ofnanomedicine in the field of molecular imaging and targeted drug delivery.We have developed a novel biocompatible nanoparticle platform using exclusively protein material. For that purpose,a recombinant protein with superior hydrophilic characteristics provided by the amino acid repeat proline, alanine,serine and lysine (PASK) was designed and crosslinked into nanoparticles with polyglutamic acid (E) using themesoporous silica template technology. The obtained PASKE particles were labelled with near infrared fluorescentmolecules (Cy7) and functionalized with an anti P-Selectin antibody (VH10) via click chemistry to provide specifictargeting of inflamed vessels and thrombosis.The low fouling properties were tested with a cell association assay using human monocytic cells (THP-1) and murinemacrophages (RAW 264.7) and the PASKE particles were insignificantly phagocytosed compared to silica particles(3±1% Vs 60±7%, p<0.001 with THP-1 cells and 5±1% Vs 61±12%, p<0.001 with RAW 264.7 cells). Tissuebiodistribution studied with a near infrared imaging system revealed that the PASKE particles preferentiallyaccumulate in the reticuloendothelial system and are cleared via the kidney. Histology examinations indicated that fulldegradation of particles occurs in the spleen within 24 hours. The Cy7 labelled PASKE particles functionalized withanti-P-Selectin (PASKE-Cy7-VH10) were tested in vivo in a carotid thrombosis mouse model and near infraredconfocal microscope observation confirmed their accumulation in the thrombotic artery compared to non-functionalized control (PASKE-Cy7).These novel, fully protein based PASKE particles have promising targeting capacities to thrombosis, strong signal innear infrared optical imaging coupled with low fouling and biodegradable features essential for clinical translation.Future work will consist in validating their use as a molecular imaging tool of vascular diseases with clinicalintravascular near-infrared fluorescence systems.

IMAGE CAPTION:

SESSION TITLE: Scientific Session 15: Basic Biology - Imaging BiochemistryCONTROL ID: 2735824TITLE: Non-Invasive Quantification of Spatiotemporal Oxidative Stress after Acute Kidney InjuryPRESENTER: Neal ParagasAUTHORS (FIRST NAME, LAST NAME): Tomoaki Miyazaki2, Toni A. Hsu2, Pavlo Khodakivskyi3, Kevin P. Francis1,Elena Dubikovskaya3, Neal Paragas2

INSTITUTIONS (ALL): 1. Life Sciences, PerkinElmer, Hopkinton, MA, United States. 2. Medicine, University of Washington, Seattle, WA, United States. 3. Swiss Federal Institute of Technology of Lausanne, Lausanne, Switzerland.ABSTRACT BODY: Abstract Body: We report a cell-specific in vivo bioluminescent imaging methodology to monitor ROS dynamics as aprognostic biomarker for acute kidney injury (AKI). We dynamically measured hydrogen peroxide (H2O2) generation,after AKI using a set of transgenic animals engineered with renal cell-specific luciferase reporters in combination witha hydrogen peroxide caged-luciferin (HPC-Luciferin). With this approach, we discovered that the antioxidantMitoTEMPO significantly reduced oxidative stress. Tissue specific Rosa26-luciferase reporters were activated through Cre-mediated recombination in four compartmentsof the kidney – glomerulus (PodR26Luc/+), proximal tubule (SLC34a1R26Luc/+), collecting duct (HoxB7R26Luc/+)and endothelium (Cdh5R26Luc/+) – along with a global (EIIaR26Luc/+) reporter were bred. Recombination wasconfirmed in these reporter animals by imaging luciferase activity with a saturating dose of D-Luciferin (150 mg/kg).Bioluminescence emanating from these specific reporter cells was collected in vivo by an optical imaging system. Luciferase reporter mice were challenged with 30 min bilateral IRI, and whole body optical images were collected 3, 6,12 and 24 h using both a D-luciferin and a H2O2 sensitive caged-luciferin. In the presence of endogenous H2O2production, this hydrogen peroxide caged-luciferin (HPC-luciferin), comprised of a self-immolative benzylic linkerfusing an aryl boronic acid to firefly luciferin boronic acid benzyl ether, is cleaved to liberate free intracellular D-Luciferin. Once D-Luciferin is freed, it is catalyzed by intracellular luciferase in a two-step oxidative reaction, enablingbioluminescence. EIIaR26Luc/+ animals revealed a significant increase in H2O2 reporter activity in both kidneys 6 h after IRI challenge.HoxB7R26Luc/+ and CDH5R26Luc/+ reporter activity mirrored HPC-luciferin activation in the global EIIaR26Luc/+

reporter animal. Surprisingly, SLC34a1R26Luc/+ reporter animals did not have significant change in H2O2 activityafter IRI, while PodR26Luc/+ was consistent with histological evidence that glomerular epithelia are not injured in AKI.In vivo measures of EIIaR26Luc/+ and HoxB7R26Luc/+ HPC-luciferin activity was in agreement with lipid peroxidation(MDA) levels, with all peaking at 6 h after IRI. Mitochondria are a prominent source of cellular ROS in the cell and thus a critical driver of IRI. MitoTEMPO, amitochondria-specific superoxide scavenger, was used to investigate of the role of mitochondrial ROS in the OSresponse to IRI in the collecting duct. An i.p. bolus of MitoTempo (10mg/kg) administered immediately prior to IRIsurgery, led to a significant reduction in H2O2 activity at 6 h and overall delay in upregulation of ROS in the collectingduct. Using HPC-luciferin, we could monitor the generation of the ROS in real-time after IRI, and we found that kidney lipidperoxidation is dependent on ROS. Since the role of sustained inflammation and OS have been demonstrated inexperimental models of renal fibrosis our model provides valuable insight into the earliest mechanisms of the pro-fibrotic pathway activation and a method to test for therapeutic intervention.(No Image Selected)

SESSION TITLE: Scientific Session 19: Basic Biology - Cell Imaging & BioengineeringCONTROL ID: 2732695TITLE: Evaluation of the Rat Pharmacokinetics of 89Zr-GSK3128349, a novel Albumin-Binding Domain Antibody(AlbudAb), for Immuno-PET Imaging in the Clinic PRESENTER: Hasan AlsaidAUTHORS (FIRST NAME, LAST NAME): Mary Rambo1, Ciara Seal2, Matthew Szapacs2, Danielle Vugts3, MarieDavies4, Sean Zhang4, Kevin Thorneloe4, Beat M. Jucker1, Hasan Alsaid1

INSTITUTIONS (ALL): 1. Bioimaging, PTS, GlaxoSmithKline, King of Prussia, PA, United States. 2. Exploratory Biomarkers, PTS, GlaxoSmithKline, King of Prussia, PA, United States. 3. Radiology & Nuclear Medicine, VUMC, Amsterdam, Netherlands. 4. Translational Medicine, PTS, GlaxoSmithKline, King of Prussia, PA, United States.ABSTRACT BODY: Abstract Body: Introduction:Conjugation or fusion to albumin-binding domain antibodies (AlbudAbs) is a novel approach to altering the half-lifeand/or distribution of both biological and small molecule therapeutics1. The 89Zr labeled GSK3128349 (89Zr-GSK’349) AlbudAb is being utilized in an Immuno-PET clinical imaging study2 to determine the biodistribution and PKof GSK’349 in healthy subjects. The goal of this preclinical study in rats was to determine if radiolabelling of GSK’349alters the clearance, exposure profile and half-life of GSK’349.Method:GSK’349 was reacted with N-suc-desferrioxamine-TFP ester in a 1:2 ratio and subsequently iron was removed byEDTA challenge at pH 4.2-4.5. After purification by size exclusion chromatography, N-suc-DFO-GSK’349 wasradiolabeled3 with 89Zr. All procedures were approved by the Animal Care and Use Committee of GlaxoSmithKline.Crl:WI(Han) male rats (body weight: BW~300g, n=3/group) were administered either GSK’349 or 89Zr-GSK’3490.36mg/kg (3.5MBq) via intravenous tail vein injection. Blood samples were collected before and up to 144 hours postinjection. The 89Zr-GSK’349 uptake in blood was calculated and decay corrected using Wizard 2480 Gamma Counter(PerkinElmer), and the drug concentration in the blood was calculated based on the radioactivity measurement usingthe following equations: Injected dose (mg)= BW (kg) * dose (0.36mg/kg), Drug Concentration (ng/ml)= injected dose(ng) * %ID/g / 100. Plasma samples from both groups were also analyzed to measure the PK of GSK’349 using ananalytical method based on immunocapture and trypsin digestion, followed by UHPLC-MS/MS analysisof a CDRportion of the GSK’349 molecule.Results:Fig 1 shows a similar PK profile in plasma for GSK’349 and 89Zr-GSK’349 treated groups as measured using theUHPLC-MS/MS method. The 89Zr-GSK’349 PK profile measured using the radiodetection method yielded similarresults to those obtained by UHPLC-MS/MS, as demonstrated by a significant correlation (fig 2: r=0.96, P<0.0001).The different PK parameters (tab 1) including the systemic exposure to GSK’349 from the start of dosing to the lastquantifiable time point (AUC0-t), the median time (Tmax) at which the maximum concentration (Cmax) was observed,the mean terminal elimination half-life, the mean plasma clearance, and the mean volume of distribution did not showany substantial differences between GSK’349 and 89Zr-GSK’349 groups using the UHPLC-MS/MS and theradiodetection methods.Conclusions:The clearance, exposure profile, and half-life of GSK’349 did not appear to change after labeling it with 89Zr. Hence,utilizing immuno-PET imaging with 89Zr-GSK’349 is considered a reasonable approach to estimate the biodistributionof the GSK’349 AlbudAb in humans. We also show that the radiodetection method can be used to calculate theAlbudAb drug concentration and the PK parameters of 89Zr-GSK’349 with similar accuracy to standard UHPLCMS/MS methods. Radiodetection PK methods may therefore be applied for future 89Zr labeled proteins.References1 RL O’Connor-Semmes. Clin Pharmacol Ther. 2014 Dec;96(6):704-12.2 ClinicalTrials.gov Identifier NCT02829307.3 Verel I. J Nucl Med. 2003, 44, 1271-1281.

IMAGE CAPTION: