CANCER & IMMUNOTHERAPY SYMPOSIUM2017HOSTED BY ACEA BIOSCIENCES

RENAISSANCE ARLINGTON CAPITAL VIEW HOTEL 2800 SOUTH POTOMAC AVENUEARLINGTON, VIRGINIA 22202 USA

MARCH 31 - APRIL 1, 2017 | WASHINGTON, D.C. USA

RENAISSANCE ARLINGTON CAPTITAL VIEW HOTEL2800 SOUTH POTOMAC AVENUEARLINGTON, VIRGINIA 22202 USA

Founded in 2002, ACEA Biosciences, Inc. is a privately owned biotechnology company that is pioneering the development and commercialization of high performance, cutting edge cell analysis instruments for life science research and clinical diagnostics.

Employing noninvasive impedance microelectrodes in automated high-throughput plate formats, our xCELLigence® Real-Time Cell Analysis (RTCA) instruments maximize the physiological relevance of data extracted from in vitro cellular assays. These instruments enable label-free, real-time monitoring of cell proliferation, cell size/morphology, cell-substrate attachment quality, and cell invasion/migration. By eliminating the time- and labor-intensive steps of traditional methods, RTCA vastly improves efficiency and overall productivity. Moreover, the flexibility, sensitivity, and reproducibility of RTCA make it a standout amongst cell-based assay platforms.

About ACEA Biosciences, Inc.

ACEA is also helping to revolutionize the field of flow cytometry by designing and producing high performance, customizable benchtop cytometers at accessible price points. By including features such as 15 fluorescence detection channels with 3 lasers, direct volumetric based cell counting, and a versatile high throughput walkaway autosampler, ACEA’s line of NovoCyte™ flow cytometers is raising the bar for what is expected of benchtop machines.

xCELLigence® and NovoCyte™ instruments are currently being used worldwide in academic, industrial, and hospital labs for preclinical drug discovery and development, toxicology, safety pharmacology, disease studies, clinical diagnostics, and basic research.

SYMPOSIUM FLOOR PLAN

FRIDAY(STUDIO B)

SATURDAY(SALONS 1-3)

MEETING AGENDA

8:00 AM - 9:00 AM Breakfast & Registration

9:00 AM - 9:30 AM Welcome Yama Abassi, Ph.D. Vice President, ACEA Biosciences

9:35 AM - 10:05 AMEvaluation of Novel Chimeric Antigen Receptor Constructs Using Sensitive, Real-Time Measurement Of B Cell Killing

Dan MacLeod, Ph.D. Group Leader, Cell Therapy ResesarchPrecision BioSciences, Inc.

10:10 AM - 10:40 AMDevelopment of Antigen-specific Human Tumor Cell Killing Assays for Testing of Immunotherapeutic Agents

Mike Overstreet, Ph.D. Research Scientist, MedImmune

10:40 AM - 11:00 AM Morning Break

11:00 AM - 11:30 AMTargeting the Unfolded Protein Response (UPR) Signaling Components Differentially Affects Immune Cell Activities

Katherine Cook Ph.D.Assistant Professor, Department of Surgery Wake Forest University

11:35 AM - 12:05 PM Intensifying T cell Responses to Weakly Immunogenic or Lowly Expressed Tumor Antigens

Eduardo Davila, Ph.D. Associate Professor of Microbiology & ImmunologyUniversity of Maryland School of Medicine

12:05 PM - 1:00 PM Lunch & Networking

1:05 PM - 1:35 PMHarnessing the Full Power of Impedance-Based Cytotoxicity Data through the xCELLigence Immunotherapy Software

Fabio Cerignoli, Ph.D.Senior Field Application ScientistACEA Biosciences

1:40 PM - 2:20 PM Designer T-cells, Designer Drugs, and Proof of Concept with xCELLigence

Andrew MacLean, Ph.D.Assistant Professor of Microbiology & Immunology, Tulane School of Medicine

2:25 PM - 2:55 PMImmunosuppressive Tumor-Infiltrating Myeloid Cells Mediate Adaptive Immune Resistance via a PD-1/PD-L1 Mechanism in Glioblastoma

Joey Orpilla Research Assistant, UCLA Department of Neurosurgery, Brain Tumor Program

3:00 PM - 3:15 PM Late Breaking Abstract

3:20 PM - 4:00 PM Panel Discussion

Poster Award

4:00 PM Meeting Concludes

Saurday, April 1, 2017

Friday, March 31, 2017

4:30 PM - 6:00 PM Registration

6:00 PM - 9:00 PM Welcome Reception/Poster Session

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Featured Speakers

Dr. Yama A. Abassi is Vice President of ACEA Biosciences. He received his undergraduate degree in biochemistry with honors from the State

University of New York/Stony Brook in 1992, and a Ph.D. in Molecular, Cell, and Developmental Biology from the University of California at Santa Barbara

in 1999. Dr. Abassi joined the Burnham Institute for Cancer Research as an NIH post-doctoral fellow in 1999 and spent the next few years studying oncogene signaling pathways and their role in cancer biology. Dr. Abassi joined ACEA Biosciences in 2003 to help develop a new technology for studying cells using microelectronics. Dr. Abassi’s efforts in this field have earned him numerous publications and patents and have led to the successful development and commercialization of the xCELLigence Real-Time Cell Analysis (RTCA) instrument globally. Dr. Abassi continues to pursue his research interests in developing technologies and applications for in vitro cell-based assays in different fields, including cancer immunotherapy. He also oversees several collaborations with the US EPA that focus on assessing the impact of toxic environmental compounds (in particular, compounds which may interfere with estrogen signaling pathways).

SELECTED xCELLigence PUBLICATIONS

1. Label-free, real-time monitoring of IgE-mediated mast cell activation on microelectronic cell sensor arrays. Abassi YA, Jackson JA, Zhu J, O'Connell J, Wang X, Xu X. J Immunol Methods. 2004;292(1-2):195-205.

2. Dynamic monitoring of cell adhesion and spreading on microelectronic sensor arrays. Atienza JM1, Zhu J, Wang X, Xu X, Abassi Y. J Biomol Screen. 2005;10(8):795-805.

3. Dynamic and label-free monitoring of natural killer cell cytotoxic activity using electronic cell sensor arrays. Zhu J, Wang X, Xu X, Abassi YA. J Immunol Methods. 2006;309(1-2):25-33.

4. Kinetic cell-based morphological screening: prediction of mechanism of compound action and off-target effects. Abassi YA, Xi B, Zhang W, Ye P, Kirstein SL, Gaylord MR, Feinstein SC, Wang X, Xu X. Chem Biol. 2009;16(7):712-23.

5. Functional cardiotoxicity profiling and screening using the xCELLigence RTCA Cardio System. Xi B, Wang T, Li N, Ouyang W, Zhang W, Wu J, Xu X, Wang X, Abassi YA. J Lab Autom. 2011;16(6):415-21.

6. Real-time growth kinetics measuring hormone mimicry for ToxCast chemicals in T-47D human ductal carcinoma cells. Rotroff DM, Dix DJ, Houck KA, Kavlock RJ, Knudsen TB, Martin MT, Reif DM, Richard AM, Sipes NS, Abassi YA, Jin C, Stampfl M, Judson RS. Chem Res Toxicol. 2013;26(7):1097-107.

7. An impedance-based cellular assay using human iPSC-derived cardiomyocytes to quantify modulators of cardiac contractility. Scott CW, Zhang X, Abi-Gerges N, Lamore SD, Abassi YA, Peters MF. Toxicol Sci. 2014;142(2):331-8.

Yama A. Abassi, Ph.D.Vice President ACEA Biosciences, Inc.

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Featured Speakers

Dr. MacLeod received his Ph.D. in Molecular Pathology and Biomedical Sciences from the University of California, San Diego (UCSD). He subsequently conducted

postdoctoral research at the International AIDS Vaccine Initiative Neutralizing Antibody Center at the Scripps Research Institute. Prior to joining Precision BioSciences,

Dr. MacLeod was an Investigator at GlaxoSmithKline working on immunotherapy drug discovery programs.

Dr. MacLeod is currently leading a group in Precision BioScience’s Cell Therapy Research team, focused on expanding the use of Precision’s propriety ARCUS gene-editing technology to engineer T cells for cancer therapy. Precision is developing chimeric antigen receptor (CAR) T cells derived from healthy donor cells and using our ARCUS technology to make the cells safe upon adoptive transfer into unrelated patients, with the goal of making adoptive cell therapies more consistent, scalable, and accessible to a greater number of patients.

PRESENTATION ABSTRACTEvaluation of Novel Chimeric Antigen Receptor Constructs Using Sensitive, Real-Time Measurement of B Cell Killing

Adoptive cellular therapy using CAR T cell therapies have produced significant responses in patients with CD19+ hematological malignancies. Extensive efforts are underway to improve the safety & efficacy of anti-CD19 CAR T cells, and translate these promising results to additional targets and therapeutic indications. Optimization of anti-CD19 CAR T cells & generation of new CAR T constructs requires modifications to both the extracellular binding domains and the intracellular signaling domains, and requires sensitive methods for evaluating CAR T cell function. Traditional methods for evaluating the cytotoxic activity of CAR T cells are of limited predictive value in terms of in vivo activity of a CAR T product, as these methods reflect short-term killing potential rather than robust ability to expand, persist & exhibit serial killing. We have used the xCELLigence instrument and both B Cell Killing Assays and Leukemic Cell Killing Assays to test multiple CAR constructs with modified ectodomains & endodomains, and have found this platform to be sensitive enough to detect subtle yet significant differences in performance that were not observed in traditional killing assays, and better reflect how the cells would be anticipated to function in vivo. Furthermore, the sensitivity of the instrument has enabled us to evaluate CAR constructs using cell numbers that are orders of magnitude lower than other assays, greatly enhancing our throughput and capabilities.

Dan MacLeod, Ph.D.Group Leader, Cell Therapy ResearchPrecision BioSciences, Inc.

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Featured Speakers

Michael received his Ph.D. from the Johns Hopkins University and conducted his postdoctoral studies at the University of Rochester. In

2012, he began working at Amplimmune, a small biotech focused on T cell immunotherapy, where he led early-stage pipeline projects and supported

translational studies of later-stage assets. In 2015, he moved to his current position in the BioSuperiors group at MedImmune, where he is part of a team focused on

progressing novel therapeutic approaches against clinically-validated targets and leads projects in immuno-oncology and antibody-drug conjugates.

PRESENTATION ABSTRACTDevelopment of Antigen-specific Human Tumor Cell Killing Assays for Testing of Immunotherapeutic Agents

In the study of costimulation and checkpoint inhibition in T cells, it is imperative to thoughtfully consider the types of in vitro models that will be utilized to study pathway blockade or agonism. While there is a role for polyclonal stimulation of T cells for certain studies, an artificially-strong T cell stimulus can saturate signaling machinery, rendering a T cell potentially resistant to costimulation and/or checkpoint inhibition. Thus, one must develop more physiologic models where T cell stimulus more accurately reflects that encountered in vivo, especially when conducting attempting to modify effector function. Here we discuss approaches to studying human T cell biology in vitro, using antigen-specific model systems designed to interrogate T cell killing of tumor cells. We will also begin exploring under-studied facets of T cell biology essential for the success of immunotherapy in the clinic.

Mike OverstreetResearch ScientistMedImmune

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Featured Speakers

Katherine CookResearch InstructorLombardi Comprehensive Cancer Center, Wake Forest University

Katherine L. Cook is an Assistant Professor in the Department of Surgery and member of the Comprehensive Cancer Center at Wake Forest University.

She received a B.S. degree in Biochemistry from the State University of New York at Oswego and a Ph.D. in Molecular Medicine & Translational Science from Wake

Forest University School of Medicine. She completed her postdoctoral fellowship and was a Research Instructor at Georgetown University. Dr. Cook has been active in the area of cancer therapeutics for several years. Her dissertation project investigated the role of angiotensin-(1-7), a small endogenous peptide hormone of the renin-angiotensin system, as a potential chemotherapeutic agent to treat breast cancer. She was awarded a Department of Defense (DOD) Breast Cancer Research Program (BCRP) Predoctoral Traineeship award in 2008. Her research efforts, among others, served as a basis for a Phase I clinical trial at Wake Forest University Comprehensive Cancer Center. Dr. Cook continued her professional development as a breast cancer researcher by accepting a postdoctoral fellowship in Dr. Robert Clarke’s group at Georgetown University Lombardi Comprehensive Cancer Center, which was funded by a DOD BCRP Postdoctoral Fellowship award in 2012. She has developed various different projects studying the role of autophagy and the unfolded protein response mediating anti-estrogen resistant breast cancers. Dr. Cook has collaborated on over 25 published manuscripts. Dr. Cook is currently funded by a Prevent Cancer Foundation Research Grant and an American Cancer Society Research Scholar Grant to determine dietary effects on obesity-mediated breast cancer.

PRESENTATION ABSTRACTTargeting the Unfolded Protein Response (UPR) Signaling Components Differentially Affects Immune Cell Activities

The unfolded protein response (UPR) is an endoplasmic reticulum stress pathway controlled by glucose-regulated protein-78 (GRP78), to mediate inositol-requiring enzyme-1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6. We show deletion of GRP78, IRE1, and PERK through RNAi differentially regulates macrophage polarization. Specifically, PERK inhibition enhances macrophage proliferation and macrophage-mediated killing of 4T1 breast cancer cells, but not GRP78 or IRE1 inhibition. Targeting UPR in the cancer cells also differentially affected macrophage cytolytic capacity. Tumoral IRE1 or GRP78 inhibition enhanced macrophage phagocytosis. Conditioned media from control or GRP78-silenced cancer cells indicated reciprocal regulation of CD80 and CD206; suggesting regulation of M1/M2 macrophage plasticity by secreted factors. GRP78 targeting in mice resulting in a cytokine shift and increased tumoral CD80+/CD68+ cells, suggesting a M1-like macrophage profile. Inhibition of UPR components in both macrophage and cancer cells, similar to what would be observed in systemic UPR targeting therapies, indicated that either PERK or GRP78 inhibition enhances macrophage cytolytic clearance of cancer cells. Recently, it was demonstrated that macrophage polarization can influence immune checkpoint therapy resistance. To determine whether UPR signaling effects immunotherapy resistance, matched PBMC population from melanoma patients before/after developing ipilimumab resistance indicated increased UPR signaling and an elevated M2-like macrophage population, supporting a novel role UPR signaling in immune cell regulation and anti-CTLA-4 therapy resistance.

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Featured Speakers

Dr. Eduardo Davila, received his Ph.D. degree from Mayo Clinic Graduate School where focused on developing immune adjuvants and vaccines to elicit antitumor T

cell responses. Dr. Davila is an Associate Professor of Microbiology and Immunology at the University of Maryland School of Medicine. He serves as the program leader

for the Tumor Immunology and Immunotherapy Research Program at the Greenebaum Comprehensive Cancer Center at the University of Maryland and the Director of the Science Training for Advancing Biomedical Research Postbaccalaureate Program. Dr. Davila and his research team focus on achieving two main goals. First, they aim to develop novel approaches for treating established tumors by enhancing cytotoxic T cell responses towards weakly immunogenic and lowly expressed tumor antigens (TAgs). Second, they focus on developing strategies to overcome the immunosuppressive tumor microenvironment (TME) in order to restore anti-tumor T cell responses.

PRESENTATION ABSTRACTIntensifying T cell Responses to Weakly Immunogenic or Lowly Expressed Tumor Antigens

T cell–based immunotherapies are a promising approach for patients with advanced cancers. However, various obstacles limit T cell efficacy, including suboptimal T cell receptor (TCR) activation and an immunosuppressive tumor environment. We developed a novel fusion protein that activates Toll-like receptor (TLR) signals in CD8+ T cells in a TLR ligand-independent but TCR-dependent manner resulting in enhanced responses against weakly immunogenic and/or poorly expressed tumor antigens including melanoma neoantigens. T cells engineered to express this fusion protein exhibit increased proliferation and expression of effector and co-stimulatory molecules in a tumor antigen-dependent manner. Engineered T cells show improved antitumor responses in mice and are associated with a unique tumor cytokine/chemokine signature, improved T cell infiltration and reduced markers of T cell exhaustion ex vivo. Engineered T cells further re-shaped the tumor environment by reducing the number of macrophages with an immunosuppressive phenotype and by inducing the expression of costimulatory proteins on cells. Our approach represents a unique and versatile method to help overcome immunosuppression and enhance T cell responses.

Eduardo Davila, Ph.D.Associate Professor of Microbiology & Immunology University of Maryland School of Medicine

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Featured Speakers

Dr. Fabio Cerignoli is Senior Field Application Scientist for the xCELLigence RTCA platform at ACEA Biosciences, where he is highly involved in developing new tools

and reagents and also in validating the xCELLigence technology across multiple cellular and assay models. He received his Ph.D. in Molecular Pathology from the University of

Rome "La Sapienza" in Italy and moved to San Diego to study signal transduction and stem cell differentiation at the Sanford Burnham Medical Research Institute. Before joining ACEA, Dr. Cerignoli worked with other San Diego-based Biotechnology Companies where he was awarded several NIH small business grants to develop new tolls to study compound-mediated cardiotoxicity.

PRESENTATION ABSTRACT Harnessing the Full Power of Impedance-Based Cytotoxicity Data through the xCELLigence Immunotherapy Software

The introduction of the xCELLigence RTCA platform for monitoring immune cell-mediated cytolysis is revolutionizing how cancer immunotherapy is evaluated in vitro. High sensitivity and real time kinetic are revealing the complex dynamic of the interaction between target and effector cells, but also the naïve assumption that the Cell Index parameter alone fully reflects the number of undamaged target cells.

To provide better potency evaluation, ACEA Biosciences has developed a new software suite specifically designed for immunotherapy applications and which provides cytotoxicity evaluation through normalization against a series of internal controls. Furthermore, innovative parameters that take full potential of the time component in the xCELLigence assay have been introduced to compare treatment efficacy not only at a specific time point, but through the whole duration of the experiment.

The presentation will focus on few examples of actual data and on how the new software allows a more complete evaluation of reagents efficacy.

Fabio Cerignoli, Ph.D.Senior Field Application Scientist ACEA Biosciences

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Featured Speakers

Andrew MacLean is currently an assistant professor at Tulane University School of Medicine, Tulane Center for Aging, Tulane Brain Institute, and the

Tulane National Primate Research Center. He enjoys mentoring graduate and undergraduate students while designing, building and studying cell culture models

for the blood-brain barrier. These are used to model multiple diseases, mainly infections affecting the CNS. He has been an avid user of the xCELLigence DP system since 2010, including the only inverted 3D real-time blood-brain barrier model. Beyond real time measures of cell function, he examines neuropathogenesis of infectious and behavioral diseases, focusing on BBB and glial activation. Recently, these studies have examined mechanisms of glial arbor change in healthy aging, and during disease. He currently has funding through NINDS, NIMH, NIDA and the Department of Veterans Affairs.

PRESENTATION ABSTRACTDesigner T-cells, Designer Drugs, and Proof of Concept with xCELLigence

The MacLean lab purchased the xCELLigence RTCA DP in 2010. Studies using CIM plates allowed us to develop an inverted real time 3D blood-brain barrier (BBB) model. This model has been used, so far, for HIV studies, primarily showing how SIV-infected macrophages impact BBB permeability. Combined with studies using E plates, we have examined how endothelial cells are differentially activated in a polarized manner.

We have also used E plates for a number of studies linked to immunotherapy. We redirected cytotoxic T cells toward HIV. We developed a novel assay that directly measures cytotoxicity in real time using xCELLigence DP system. CD4-Chimeric Antigen Receptor or control transduced populations were added to HEK 293T cells expressing Env (or control) at effector:target ratio of 1:1 and the change in the electrical impedance compared with baseline impedance was used to measure the Env-specific cytotoxicity. The CD4-CAR-transduced rhesus T cells specifically killed Env+ target cells.

Beyond this published study, we have used the DP system to demonstrate decreased trafficking of cells following antibody therapy of the host. Both immunotherapy studies will be discussed.

Andrew MacLean, Ph.D.Assistant Professor of Microbiology & Immunology Tulane School of Medicine

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Featured Speakers

Joey Orpilla is a research associate in the laboratory of Dr. Robert Prins as a part of the UCLA Department of Neurosurgery Brain Tumor Program. He graduated

with honors from UCLA in Molecular, Cell and Developmental Biology, with focuses in biomedical research and bioinformatics. He has utilized the xCELLigence RTCA

system for 4 years, which has resulted in several publications that have contributed to the advancement of immunotherapy research in brain tumors.

PRESENTATION ABSTRACTImmunosuppressive Tumor-Infiltrating Myeloid Cells Mediate Adaptive Immune Resistance via a PD-1/PD-L1 Mechanism in Glioblastoma

Adaptive immune resistance in the tumor microenvironment appears to attenuate the immunotherapeutic targeting of glioblastoma (GBM). In this study, we identified a tumor-infiltrating myeloid (TIM) cell population that expands in response to DC vaccine treatment. The aim of this study was to understand how this PD-L1-expressing population restricts activation and tumor-cytolytic function of vaccine-induced tumor infiltrating lymphocytes (TILs).

Joey OrpillaResearch AssistantUCLA Department of Neurosurgery, Brain Tumor Program

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Featured SpeakersPoster AbstractsPOSTER #1

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Akwasi AsamoahDepartment of Wood Science and Technology, Kwame Nkrumah University of Science and Technology, University Post Office, Kumasi

Plants and animals are often outwardly seen to be so dissimilar, but they are both with cell membrane which happens to be the one that gives birth to plant cell walls. Nevertheless, it is DNA which controls the formation of both plant and animal cells of all living organisms and even viruses which are known to be non-living outside living cells. Thus, for both types of cells to take instructions from DNA and its RNA copies, both types of cells must be assembled together similar ways. Both types of cells must hold similar secrets and innovations in their assemblage which when well learned could be useful for the advancement of various natural fields of endeavor. The plant cell for instance being the most basic unit of the earliest life form holds secrets and innovations which could reorient the thinking and processes of practitioners in the biomedical field in ways which may not have been yet imagined. Cellulose is the structural component in plant cell walls, tunicate mantles and mollusk exoskeleton. It is no mistake that its has survived the years of evolution to be the most abundant biopolymer in the biosphere. In whichever matrices or composites that it finds itself, cellulose mixes and binds seamlessly well with other chemical and structural components such as protein, DNA, RNA, fats and oils which makes it an unmatched template or platform to this day. This must be so because cellulose exhibits similar chemistry and organization with DNA, RNA, fats and oils and their semblances at relevant length scales which might not be well highlight in the literature. Thus, this presentation seeks to highlight these similarities and the accompanying opportunities which such appreciation may offer to the biomedical fields especially in the areas of virus and cancer treatments.

What Opportunities Does Nanocellulose and Protein Assemblies Offer for Cancer Treatment?

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Poster Abstracts

Fabio Cerignoli, Biao Xi, Garret Guenther, Lincoln Muir, Leyna Zhao, Yama AbassiACEA Biosciences Inc., San Diego, CA

In vitro characterization of reagents efficacy in the context of cancer immunotherapy is a necessary step before moving to more expensive animal models and clinical studies. However, current in use in vitro assays like Chromium-51, ATP-based luminescence or flow cytometry are either difficult to implement in high throughput environment or are mainly based on end point methodologies that are unable to capture the full dynamic of the immune response. Here we present the adaptation of an impedance-based platform to monitoring cytotoxic activity of immune cells activated trough different means. Impedance technology detects cell death and proliferation of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. We utilized adherent and B cell leukemia/lymphoma cell lines as well as primary tumor cells as in vitro models for immunotherapy reagents evaluation. We seeded the cells on electrodes coated 96-well plates and monitored cell adhesion and proliferation for 24 hours. The following day effector cells were added at multiple effector:target ratios in presence of BiTEs antibodies and/or anti PD-1/PD-L1 antibodies. Impedance signal was monitored for up to seven days. Control wells were set up with effector cells only or with target plus effector cells but without antibodies. We adapted such adhesion-based technology to monitor non-adherent B-leukemia/lymphoma cells, by developing a strategy where the wells are coated with an anti-CD40 antibody. The coating allows specific adhesion and retention of B cells and measurement of changes in impedance that are proportional to cell number. Using increasing concentrations of EpCAM/CD3 BiTE we demonstrated the suitability of such impedance-based approach to quantitatively monitor the efficacy of immune cells-mediated cancer cell killing both under different effector:target ratios and antibody concentrations. Combination treatments with checkpoint reduced timing and increased amount of killed cancer cells. Similar results were also obtained with engineered Car-T cells against CD19 or NK cell lines, we were able to demonstrate specific killing of tumor B cells at very low effector:target ratios. The results were also confirmed by flow cytometry. Overall, our results demonstrate the value of such approach in measuring the cytotoxic response across the temporal scale, an aspect that is otherwise very difficult to assess with more canonical end point assays. Furthermore, the availability of 384-wells format and minimal sample handling place the technology in an ideal spot for applications in large reagent validation screening or personalized medicine, like therapeutic protocol validation directly on patient samples.

Dynamic Monitoring of Immune Response and Reagent Efficacy Through High Throughput Label-free Impedance-based Technology

Fabio Cerignoli, Biao Xi, Brandon Lamarche, Leyna Zhao, Yama AbassiACEA Biosciences Inc., San Diego, CA

In vitro monitoring cancer cells response to treatment often involves laborious sample processing and collects single data points that are against the dynamic nature of cancer cells. Here we present the adaptation of an impedance-based methodology to dynamically monitor cancer cell behavior and therapeutic response. The technology detects cell death, proliferation or migration by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. It avoids sample labeling and processing and allows continuous monitoring of cell response. Our data shows validation with cancer cell lines and primary cells monitored for cell invasion/migration, anticancer drug response and receptor signaling activation, with similar results over end point assays. Easy experiment set up and minimal sample processing make the technology ideal for applications in large screening campaigns, while label-free technology allows further analysis on the same samples through orthogonal assays.

Validation of Label-free Impedance Analysis as a Versatile Tool For Cancer Molecular Therapeutics Screening

POSTER #3

POSTER #2

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Featured SpeakersPoster AbstractsPOSTER #4

POSTER #5

Yasmin Leshem, James O'Brien, Yoram Reiter and Ira Pastan

SS1P consists of an anti-mesothelin Fv attached to Pseudomonas exotoxin A and kills tumor cells by inhibiting protein synthesis. Because of its unique mechanism of cell killing, SS1P does not inhibit the immune system like other anti-cancer drugs. Here we determined if SS1P can cooperate with anti-CTLA-4 to induce anti-tumor immunity and cause complete tumor regressions.A BALB/c breast cancer cell line was transfected with human mesothelin (66C14-M) and implanted in two different locations. SS1P was injected directly into one tumor and anti-CTLA-4 given IP.

In mice treated with anti-CTLA-4 and SS1P, tumor regressions occurred in 11 out of 12 SS1P injected tumors (91%) and in 7 out of 12 un-injected tumors (53%). No tumor regressions occurred with drugs given separately. Pathological evaluation showed a large sterile central abscess in injected tumors surrounded by a collar of inflammation containing a mixture of lymphocytes and other mononuclear cells. Analysis of responding tumors showed a time dependent increase in CD8+ cells located mainly in the inflammation collar. RNA analysis of responding tumors showed that 385/768 immune related gene transcripts were elevated by twofold or more including CD8a, PDL2 and IL1 alpha (13, 21 and 27-fold).

Altogether, combining local SS1P with anti-CTLA-4 promotes massive inflammation that cures multi-centered cancer disease providing a strong rational to explore this combination therapy in patients.

Synergistic Anti-tumor Activity of Local SS1P with Systemic CTLA-4 Blockade Causes Complete Regression of Both Injected and Un-injected Tumors in Mice

Christina Giannouli, Saurav Majumder, Richard L. Proia

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that activates five G protein-coupled receptors designated S1P-receptors type 1-5 (S1P1-5), that transmit extracellular signals into cells, and it modulates intracellular signaling as a cofactor. S1P regulates multiple cellular processes, including immune cell trafficking. Numerous studies have focused on the S1P regulation of lymphocyte trafficking, since the in vivo S1P gradients have been found to be recognized by its respective receptors and thus facilitate lymphocyte entry into circulation. Evidence from pharmacological studies suggests that S1P receptors may also be involved in innate immune cell trafficking as well. However, the role of S1P receptors on cells of myeloid origin such as monocytes and neutrophils in vivo is less clear, especially under inflammatory conditions.

In this study, we investigate the role of S1P and its receptors under septic conditions by employing the cecal ligation and puncture (CLP) model. Therefore, sepsis is induced in mice that do not express S1P1 receptor in their myeloid lineage cells, as well as in S1P-less mice, which have almost undetectable levels of S1P in their blood and lymph. The bone marrow and blood neutrophil population is increased in the S1P-less mice, suggesting that originally the S1P gradient withholds neutrophil numbers in sepsis. We have also found that the S1P1 conditional KO mice are more susceptible in sepsis. They have lower survival rate and sepsis occurs faster with mice having more bacteria growing in their blood. We have found statistical significant differences in neutrophil and monocyte populations in the blood but not in the bone marrow or other lymphoid organs, suggesting that S1P1 may be involved in the egress of these cells in sepsis. A possible role of S1P receptor role in granulopoiesis or pathogen killing is being studied as well. Collectively, the S1P-S1P1 receptor axis seems to be important for the evolvement of sepsis and the role of S1P receptors in neutrophil activation properties are under investigation.

The Role of S1P and S1P Receptors in Sepsis

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Poster Abstracts

POSTER #6

Matthew McCord, Amelie Vezina, Mark Gilbert, Sadhana JacksonDepartment of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506

IntroductionEfforts to transiently increase blood brain barrier permeability would facilitate improved CNS delivery of chemotherapy agents. Previous studies demonstrated that vascular endothelial factor (VEGF) increases permeability in traumatic brain injury and ischemic brain disease. Exogenously administered VEGF increases barrier permeability in the non-diseased CNS. In our study, we investigated the role for exogenous VEGF therapy to modulate blood-brain barrier permeability

Methods For in vitro studies, confluent mouse brain endothelial cells (BEND.3) were treated with VEGF165. Pericellular electrical impedance changes and immunofluorescence were used to assess qualitative expression of tight and adherens junction proteins at key time points. Western blotting was done to evaluate the effect of VEGF on expression of proteins associated with tight and adherens junctions. Immunohistochemistry (IHC) and transmission electron microscopy (TEM) were performed on mouse tissue samples.

Results Confluent endothelial cells treated with rVEGF165 (200ng/mL) evaluated show a significant drop in cell impedance approximately 6 hours post treatment, compared to control (more than 10-fold) reflecting increased permeability of the monolayer. Treatment with progressively higher doses of VEGF (10, 20, 50, 100, and 200ng/mL) showed a progressively greater drop in cell impedance. Subsequent treatment with bevacizumab appears to reverse the permeability affect. Immunofluorescence of endothelial cells treated with rVEGF165 (200ng/mL) shows disassembly of both tight junctions and adherens junctions between 4 and 8 hours post-treatment compared to controls. Western blots demonstrate that rVEGF165 treatment induces down-regulation of genes associated with tight junctions, such as Claudin5 and VE cadherin. TEM results also suggest VEGF-mediated blood-brain barrier modulation.

Conclusion Our results suggest that VEGF is capable of inducing transient breakdown of the blood-brain barrier, which may facilitate improved CNS delivery of chemotherapeutic agents. Our in vitro data warrants further investigations of the applicability of VEGF therapy to induce endothelial cell permeability in animal tumor models, potentially improving delivery of chemotherapeutic drugs.

Time Dependent Blood-brain Barrier Modulation by Vascular Endothelial Growth Factor to Improve CNS Drug Delivery

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Featured SpeakersPoster AbstractsPOSTER #7

Yismeilin R. Feliz Mosquea, David R. Soto Pantoja, Adam Wilson, Pierre L. Triozzi, Katherine L. CookWake Forest University, Winston Salem, NC

A critical point in cancer progression is evading recognition by the immune system. Cancer cells accomplish this by stimulating immune checkpoint signals on effector T-cells. In patients with advanced melanoma treated with immune checkpoint inhibitors, 3-year survival increased by 20%. While immune checkpoint therapies are the new first treatment option for advanced melanoma in over a decade, their efficacy is limited because resistance often develops. Understanding the molecular mechanisms of immune checkpoint inhibitor resistance is critical to develop combinatorial drug therapy to potentiate therapeutic responsiveness. The unfolded protein response (UPR) is an endoplasmic reticulum (ER) stress pathway activated when unfolded/misfolded proteins accumulate within the ER. Highly secretory cell types, such as T-cells, have larger ER cell compartments and elevated UPR components to deal with the increased protein synthesis/folding required by these cell types. Therefore, these cell types may be highly sensitive to ER stress. Our data demonstrates elevated UPR signaling components as a driver of T-cell exhaustion/dysfunction. Using a previously established T-cell exhaustion protocol, we stimulated naïve T-cells with antibodies to CD3/CD28 for 5 days and co-cultured them with MDA-MB-231 breast cancer cells, Mun2b melanoma cell line, or CMI patient-derived melanoma cell line. Exhausted T-cells displayed an increased PD-1 and PERK expression, suggesting that UPR signaling is activated during T-cell exhaustion. Treatment of naïve T-cells with DTT, a chemical agent that stimulates ER stress, also induced PD-1 and PERK compared with vehicle-treated T-cells. Gene expression analysis of T-cells indicate that co-culture with cancer cells, not CD3/CD28 activation, elevates T-cell UPR gene expression. Furthermore, induction of ER stress through low-dose DTT treatment decreased cytotoxic T-cell mediated cancer cell death, further supporting our hypothesis of ER stress inducing T-cell exhaustion. Inhibition of PERK by RNAi in TALL-104, a human cytotoxic T-cell line, enhanced T-cell mediated cancer cell clearance when exposed to ER stress-inducing agents, suggesting that PERK may represent a novel target to prevent T-cell exhaustion or restore T-cell effector capabilities. PERK inhibition in the patient-derived melanoma cells did not negatively affect T-cell-mediated killing, suggesting that systemic PERK inhibition may be an effective therapeutic strategy to enhance anti-tumor immune responses. Matched PBMC from melanoma patients before treatment or after ipilimumab therapy resistance indicated increased UPR signaling components in PBMC samples from patients after ipilimumab resistance when compared with PBMC samples before therapy, supporting a novel role UPR signaling in anti-CTLA4 therapy resistance.

UPR Signaling Promotes T-cell Dysfunction to Prevent Immune-mediated Cancer Cell Killing and Immune Checkpoint Therapy Resistance

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Poster Abstracts

POSTER #8

Eusebio Pires1,3, Austin Herr1, Kiley Anderson1, Ken Klotz1, Arabinda Mandal1, Marisa Needham1, Hui Li2, Ryan D’Souza1, Mark Stoler2, Amir Jazaeri3, David Bruns2, Charles Flickinger1 and John Herr1

Departments of Cell Biology1, Pathology2 and Obstetrics and Gynecology3 at the School of Medicine, University of Virginia, Charlottesville, Virginia, USA

SAS1B (Ovastacin/ASTL) is an oocyte specific matrix metalloprotease. The goal was to examine the transcripts and proteins in uterine tumors and test SAS1B antibodies, including a prototype immunotoxin (Saporin), for their effect in vitro on growth and viability. Unique gene-specific primers were designed. An MMMT-derived cell line, SNU539, was used for cell-based experiments. A rabbit SAS1B-polyclonal antibody generated was used to examine: 1) internalization of cell-surface SAS1B 2) antibody-dependent cell cytotoxicity and 3) immunotoxin effects on tumor growth and viability. SAS1B was found at message or protein levels in 77% of uterine tumors, with the incidence higher in MMMT tumors (87%, N=16) than endometrioid carcinomas (74%, N=59). SAS1B showed cell surface localization in live cells by immunofluorescence. Incubating SNU539 cells with SAS1B antibodies caused a transformation from a regular polygonal to rounded cells with redistributed actin cytoskeletons and shrinkage in diameter. When SNU539 cells were exposed to antibody under cold conditions followed by warming, antibody-SAS1B complexes were internalized into the cell cytoplasm where they co-localized with endocytic pathway markers EEA1/LAMP1. In a real-time cell-based assay using the xCELLigence system, tumor cells were killed in the presence of SAS1B-antibody and active complement. Inhibition of tumor cell growth and division followed by cell death occurred in culture over 72 hours using a complex of SAS1B primary antibody and secondary immunotoxin conjugate. SAS1B offers a candidate cell-surface target for development of a therapeutic tumor-selective antibody that is directed only to the tumor and an expendable population of mature oocytes thereby potentially reducing off target effects.

Immunotoxin to Cell Surface Associated Oocyte-SAS1B (Ovastacin) Protein Kills Uterine Cancer Cells

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Featured SpeakersPoster AbstractsPOSTER #9

Masaaki Tamura, Alejandro Zulbaran, Kelsey Monson, Susumu Ishiguro, Atsushi Kawabata, Deepthi Uppalapati, Naomi OhtaDepartment of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506

Although the gemcitabine is an effective chemotherapeutic agent for pancreatic cancer, unfavorable side effects often accompany. Since we have previously discovered that PKCγ C1B domain peptides effectively control tumor growth without any side effect (Kawabata et. al., Cancer Biol Ther, 2012), we sought to examine the efficacy of co-treatment with this peptide and a low dose of gemcitabine on the growth of pancreatic cancer. Although individual and co-treatment with C1B5 peptide (1µM) and gemcitabine (20nM) slightly inhibited growth of PAN02 murine pancreatic acinar cell carcinoma in 2D culture, both treatments effectively attenuated spheroid growth of PAN02 cells in 3D culture with 48.2% and 35.8% inhibition, respectively. Combination treatment with C1B5 peptide and gemcitabine further attenuated the growth of PAN02 spheroids with 69.5% inhibition. In mice bearing peritoneal allograft tumors of PAN02 cells (2.5 x 105 cells/mouse), combination treatment with C1B5 peptide at 20 mg/kg every other day and gemcitabine 15mg/kg every three days markedly inhibited tumor growth of PAN02 allografts (94% inhibition) more than individual treatment with gemcitabine (76% inhibition) or C1B5 peptide (39% inhibition). The extent of tumor growth inhibition from this combination treatment was similar to that from a higher dose (60mg/kg) treatment of gemcitabine alone. Peritoneal cavity infiltrated neutrophils and granzyme B+ lymphocyte numbers were significantly higher in the combination treatment group than in the control group. In a cell culture study, treatment with C1B5 peptide alone (1µM) significantly increased granzyme B mRNA level in Jurkat cells at 12 and 24 hours post-treatment. Simultaneous analysis of cytokine gene expressions in Jurkat cells revealed that C1B5 peptide alone significantly increased INF-γ, IL-2, and TNF-α mRNA levels, suggesting that C1B5 peptide directly stimulated Jurkat cell activation. These studies suggest that stimulation of leucocyte migration toward cancer tissues and activation of cytotoxic T cells may play important roles in tumor growth attenuation by the combination treatment of C1B5 peptide and gemcitabine. Taken together, the current study suggests that C1B5 peptide offers an effective combination treatment strategy to reduce side effects associated with gemcitabine without losing the tumoricidal effect of this agent. This work is supported in part by Kansas State University Johnson Cancer Research Center, NIH grants P20 GM103418, and Kansas Bioscience Authority Collaborative Cancer Research grant.

Co-treatment With a C1B5 Domain Peptide of Protein Kinase Cγ and a Low Dose of Gemcitabine Effectively Inhibited Pancreatic Cancer Growth in Mouse Peritoneal Cavity

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Poster Abstracts

POSTER #10

Michael G. ToveyBiomonitor SAS, Paris, France

The activity of a number of therapeutic antibodies is mediated in part by antibody-dependent cell-mediated cytotoxicity (ADCC). Traditional methods for quantifying ADCC activity are labor intensive and have a high level of inherent variability due in part to the use of primary human NK-cells from different donors as the effector cells. Although these limitations have been partially addressed by the use of an engineered effector cell line expressing the low affinity Fc receptor, FcγRIIIa (CD16), that responds to ligation of the Fc moiety of antibody bound to the specific antigen expressed on target cells by activation of a NF-AT responsive reporter gene nevertheless there is a need for an improved ADCC assay. A novel recombinant effector cell line has been developed in which the firefly luciferase reporter gene is regulated by a synthetic chimeric promoter that confers improved sensitivity, an improved dynamic range, an improved tolerance to human serum and a reduced incubation time, relative to engineered effector cell lines that express a NF-AT regulated reporter-gene, when used in an ADCC assay together with engineered target cells. Novel target cells that express a specific antigen, together with homologous control cells that do not express the antigen, have been developed for the quantification of the ADCC activity of rituximab, trastuzumab. cetuximab and TNFα antagonists.

A Novel for Improved Quantification of ADCC Activity

Amelie Vezina, Matthew McCord, Mark Gilbert, Sadhana Jackson

Drug entry into the central nervous system (CNS) is regulated by the blood-brain barrier (BBB). The BBB is poorly permeable due to lack of fenestrations, presence of endothelial cell tight and adherens junctions and multidrug resistance proteins (MRP). Despite the BBB being a major obstacle to effective drug delivery to the CNS, research on the use of vasoactive mediators to impact these junctional and and multidrug resistance proteins remains limited. Yet, Regadenoson, an FDA approved adenosine A2A receptor (A2AR) agonist, increased CNS entry of Dextran and Temozolomide in non-tumor-bearing rodents 30 minutes after administration, showing that agonism of A2AR increases BBB permeability. However, mechanisms and time of action post Regadenoson remain poorly understood. We suggest that time-dependent A2AR agonism disrupts brain endothelial cell-cell junctional interactions and MRP function to ultimately increase drug entry via paracellular transport. Mouse brain endothelial cells (bEnd.3), were treated with 10μM Regadenoson and analyzed at varied time points. Expression and permeability studies of junctional proteins (Claudin-5, ZO-1, VE-Cadherin) and MRP (ABCG2) were assessed by immunoblotting of subcellular fractions, immunofluorescence and electrical impedance measurement by the xCELLigence system. Four hours after treatment, Regadenoson increased expression of Claudin-5 in the cytosol of endothelial cells while it decreased transiently in the cytoskeletal fraction. Adherens junction protein, VE-Cadherin, expression decreased in the cytoskeletal fraction by 8 hours. Immunofluorescence staining showed transient membrane disorganization of VE-Cadherin and ZO-1 by 4 hours. Regadenoson also decreased ABCG2 expression after 1 hour, persisting for 7 hours. Lastly, Regadenoson rapidly decreased electrical cell impedance by 55% with rebound by 4 hours, suggesting an increase in permeability compared to vehicle treated cells. Our results demonstrate preliminary evidence that A2AR agonism causes transient changes in endothelial tight and adherens junctional interaction and organization with rapid diminished ABCG2 expression, which can further impair BBB efflux transport of essential CNS therapies. These studies are meaningful findings in the field of BBB dynamics. The impact of Regadenoson on BBB integrity may serve an important tool in clinical applications to enhance CNS drug delivery and improve overall disease prognosis.

Transient Modulation of the Blood-brain Barrier With Time Dependent Adenosine Receptor Agonism

POSTER #11

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