Preclinical Validation Research Articles

Preclinical Validation of a Novel Device Designed to Reduce Biofilms on Percutaneous Osseointegrated Abutments.

To assess the efficacy of a prototype cleaning device in removing biofilm from skin-penetrating abutments. Adverse skin reactions around percutaneous osseointegrated implants have been linked to poor hygiene and infection. A cleaning device made from open-cell polyfoam has been developed to facilitate manual cleaning of abutments by the patient. Serratia biofilm was grown on clean and sterile conical bone-anchored hearing system abutments including both pre-2007 (straight sided) and post-2007 (conical-sided) designs. Eighteen were photographed and then subjected to cleaning using a toothbrush or the cleaning device soaked in water or a proprietary mouthwash. Biofilm on a further group of 10 conical abutments was recorded using a scanning electron microscope before and after cleaning with the test device soaked in water. Quantitative analysis of the efficiency of the cleaning was made using image analysis. Removal of biofilm using a dry or wet toothbrush was not as effective as cleaning with the device. In 10 cases subjected to image analysis, approximately 90% of the biofilm was removed from the top third of the abutments, 85% from the middle third, and 48% from the basal third. The cleaning device is effective in removing most biofilm from the test abutments and represents a significant improvement in comparison with traditional methods such as a toothbrush.

Long-Term Sheep Implantation of WIMAGINE®, a Wireless 64-Channel Electrocorticogram Recorder.

This article deals with the long-term preclinical validation of WIMAGINE® (Wireless Implantable Multi-channel Acquisition system for Generic Interface with Neurons), a 64-channel wireless implantable recorder that measures the electrical activity at the cortical surface (electrocorticography, ECoG). The WIMAGINE® implant was designed for chronic wireless neuronal signal acquisition, to be used e.g., as an intracranial Brain–Computer Interface (BCI) for severely motor-impaired patients. Due to the size and shape of WIMAGINE®, sheep appeared to be the best animal model on which to carry out long-term in vivo validation. The devices were implanted in two sheep for a follow-up period of 10 months, including idle state cortical recordings and Somato-Sensory Evoked Potential (SSEP) sessions. ECoG and SSEP demonstrated relatively stable behavior during the 10-month observation period. Information recorded from the SensoriMotor Cortex (SMC) showed an SSEP phase reversal, indicating the cortical site of the sensorimotor activity was retained after 10 months of contact. Based on weekly recordings of raw ECoG signals, the effective bandwidth was in the range of 230 Hz for both animals and remarkably stable over time, meaning preservation of the high frequency bands valuable for decoding of the brain activity using BCIs. The power spectral density (in dB/Hz), on a log scale, was of the order of 2.2, –4.5 and –18 for the frequency bands (10–40), (40–100), and (100–200) Hz, respectively. The outcome of this preclinical work is the first long-term in vivo validation of the WIMAGINE® implant, highlighting its ability to record the brain electrical activity through the dura mater and to send wireless digitized data to the external base station. Apart from local adhesion of the dura to the skull, the neurosurgeon did not face any difficulty in the implantation of the WIMAGINE® device and post-mortem analysis of the brain revealed no side effect related to the implantation. We also report on the reliability of the system; including the implantable device, the antennas module and the external base station.

Impact of nanosystems in Staphylococcus aureus biofilms treatment.

ABSTRACT Staphylococcus aureus (S. aureus) is considered by the World Health Organization as a high priority pathogen for which new therapies are needed. This is particularly important for biofilm implant-associated infections once the only available treatment option implies a surgical procedure combined with antibiotic therapy. Consequently, these infections represent an economic burden for Healthcare Systems. A new strategy has emerged to tackle this problem: for small bugs, small particles. Here, we describe how nanotechnology-based systems have been studied to treat S. aureus biofilms. Their features, drawbacks and potentialities to impact the treatment of these infections are highlighted. Furthermore, we also outline biofilm models and assays required for preclinical validation of those nanosystems to smooth the process of clinical translation.

28. KARYOMAPPING USING WHOLE GENOME SEQUENCING DATA: PRECLINICAL VALIDATION OF A PERSONALISED APPROACH TO PGT-M

Introduction Karyomapping utilises microarray technology to track informative single nucleotide polymorphisms (SNPs) and assign haplotypes to embryos without the need for direct mutation detection (Handyside et al. 2010). Although suitable for most couples undergoing PGT-M, the use of a fixed set of SNPs may provide inadequate information in certain genetic regions or in consanguineous couples (Konstantinidis et al. 2015). Whole genome sequencing (WGS) has the potential to individualise informative SNP detection and perform concurrent direct mutation detection. However, no studies have evaluated the potential for application of the karyomapping algorithm to WGS data. Material and Methods The Platinum Genomes dataset ( https://sapac.illumina.com/platinumgenomes.html ) was analysed as a model for the application of karyomapping to WGS data. Variant call format (VCF) files were analysed according to the principles of karyomapping to create a map of informative biallelic SNPs in each grandparental haplotype. A model of embryo biopsy was created by diluting DNA from one of the family members (NA12878) to 30pg and whole genome amplification was performed with the Repli-G single cell kit (Qiagen, Hilden, Germany) as a model of trophectoderm biopsy. Whole genome sequencing was performed in triplicate on a high output NextSeq 2500 run (Illumina, California, USA) at average 15x coverage and the number of informative SNPs detected compared to the karyomapping SNP microarray. Comparison of variant calling methods was performed using the Genome Analysis Toolkit v3.4 (Broad Institute, Massachusetts, USA) and DeepVariant (Google, California, USA). Results Analysis of WGS data from the Platinum Genomes family revealed a total of 2,338,158 informative SNPs distributed evenly between grandparental haplotypes, of which 85,053 (3.6%) would have been detectable using the karyomapping SNP array. Analysis of the karyomapping window around common genes requiring PGT-M demonstrated an average 32x increase in the number of targets available for analysis in a PGT-M cycle. Analysis of 15x coverage WGS data in a model of embryo biopsy demonstrated an average 279,851 informative SNPs detected using standard variant calling and an average 285,107 using AI-based variant calling. The median distance between informative SNPs was reduced from 26,782 nucleotides to 1,212 nucleotides when comparing WGS-based karyomapping to SNP array-based karyomapping. Conclusions This study provides preclinical validation for the application of the karyomapping algorithm to WGS data. The markedly increased number of informative SNPs will allow successful feasibility studies for all couples presenting for PGT-M. The decreased distance between informative SNPs detected in a model embryo biopsy will improve diagnostic accuracy, particularly in detecting small double crossover events and reducing the number of cycles in which inadequate informative markers have been detected in the embryo sample. Finally, the use of WGS for diagnostic purposes, especially for rare and undiagnosed genetic disease, will allow the creation of a high resolution digital karyomap for couples with an affected child and improve the transition to IVF and PGT-M.

Manufacturing and preclinical validation of CAR T cells targeting ICAM-1 for advanced thyroid cancer therapy

While the majority of thyroid cancer patients are easily treatable, those with anaplastic or poorly differentiated recurrent thyroid carcinomas have a very poor prognosis with a median survival of less than a year. Previously, we have shown a significant correlation between ICAM-1 overexpression and malignancy in thyroid cancer, and have pioneered the use of ICAM-1 targeted CAR T cells as a novel treatment modality. For clinical translation of this novel modality, we designed CAR T cells possessing micromolar rather than nanomolar affinity to ICAM-1 to avoid cytotoxicity in normal cells with basal levels of ICAM-1 expression. Herein, we report the automated process of CAR T cell manufacturing with CliniMACS Prodigy (Miltenyi Biotec) using cryopreserved peripheral blood leukocytes from apheresis collections. Using Prodigy, thawed leukopak cells were enriched for CD4+ and CD8+ T cells, subjected to double transduction using lentiviral vector, and expanded in culture for a total of 10 days with a final yield of 2–4 × 109 cells. The resulting CAR T cells were formulated for cryopreservation to be used directly for infusion into patients after thawing with no further processing. We examined cross-reactivity of CAR T cells toward both human and murine ICAM-1 and ICAM-1 expression in human and mouse tissues to demonstrate that both efficacy and on-target, off-tumor toxicity can be studied in our preclinical model. Selective anti-tumor activity in the absence of toxicity provides proof-of-concept that micromolar affinity tuned CAR T cells can be used to target tumors expressing high levels of antigen while avoiding normal tissues expressing basal levels of the same antigen. These studies support the initiation of a phase I study to evaluate the safety and potential efficacy of micromolar affinity tuned CAR T cells against newly diagnosed anaplastic and refractory or recurrent thyroid cancers.

Translational Biophotonics: From research into clinical applications

Translational medicine is—much like Biophotonics—a rapidly growing field in biomedical research, with a focus on cross-functional collaboration between researchers and clinicians. We are pleased to announce the launch of Translational Biophotonics (TBIO), an international, interdisciplinary, fully open access journal. The journal is a brand extension of Journal of Biophotonics and will provide a platform where photonic technologies as well as medicine serve as the bridge in integrating translational medical photonic research into clinical practice. While a number of journals already exist on translational medicine, these are mostly specialized on disease-specific and medical areas, such as oncology, urology and so on. It was found, though, that a translational journal is missing with a special focus on photonics in medicine. Moreover, our new journal aims at publishing significant extensions of previous biophotonic research and preclinical studies which are a prerequisite to initiate and stimulate the transition of promising photonic approaches from proof of principle to the clinic for diagnostics and therapy. Thus, the journal will meet a community need for a platform combining the two areas of photonics and medicine. Translational Biophotonics will distinguish itself also from existing journals publishing “lasers in medicine” which usually focus on single aspects like laser techniques in medical and aesthetic treatment, diagnostic applications or the fundamentals of biophotonic progress. The new journal will cover all areas—photonics and medicine—with a much wider breadth of photonic techniques and medical applications. Translation between preclinical and clinical research is the purpose of our journal, aiming at enhancing new clinical applications of biophotonics and, thus, extending the frontiers of light research into clinical applications. The constantly growing number of submissions in Journal of Biophotonics over the last 12 years has demonstrated the broad wealth of biophotonic technologies and laser applications, ranging from cutting edge spectroscopy to microscopy and tomographic imaging approaches for medical diagnosis and therapy. However, most of these contributions cease with the publication of proof-of-concept studies on, for example, phantoms, ex vivo samples or a very limited number of patients where optical and photonic technologies have proven their great potential regarding specific diagnostic and therapeutic issues. Translational Biophotonics will go beyond such proof-of-concept studies by focusing on translational preclinical studies. This stage of research starts before clinical trials on new medical devices, diagnostic tools, therapy solutions or drugs can begin in humans and when important feasibility, iterative and biocompatibility testing is conducted, and safety data are collected. Translational Biophotonics publishes original papers, reviews, letters and preclinical validation studies. Special features are commissioned tutorial reviews that are intended to help researchers and clinicians understand the common ground in their work. All articles published by Translational Biophotonics will be fully open access and immediately freely available to read, download and share. For a limited period, the usual article publication charge payable by authors or institutions will be waived. The Editors are supported by a stellar international editorial board of renowned scientists and clinicians from different research fields. Their expertise and scientific background will help to shape the future and success of this interdisciplinary journal. We look forward to working with authors, reviewers and board members to provide excellent, high-quality content from both the biophotonics and the translational medicine world. You are invited to send us your feedback and propose new topics or features and we welcome your excellent work to be published in Translational Biophotonics June 2019 The Editorial Team Jürgen Popp Editor-in-Chief Leibniz Institute of Photonic Technology and Institute for Physical Chemistry & Abbe Center of Photonics (ACP) Friedrich Schiller University Jena, Germany Carsten Philipp Editor-in-Chief Zentrum Lasermedizin Evangelische Elisabeth Klinik Berlin, Germany Ronald Sroka Editor-in-Chief Department of Urology University Hospital Munich Munich, Germany Christoph Krafft Assistant Editor Leibniz Institute of Photonic Technology Jena, Germany Regina Hagen Journal Publishing Manager Wiley Berlin, Germany

An Air-Operated Bistatic System for Breast Microwave Radar Imaging: Pre-Clinical Validation.

This study presents a breast microwave radar imaging system designed for bistatic operation in air. The system operates in the range of 1-8 GHz and employs a double-ridged horn antenna array with four degrees of freedom. A hemispherical breast phantom with a tumor inclusion of 1.5 cm diameter was used for validation. Reconstructed datasets resulted in artifact-free images where the tumor presence was detected. Signal to clutter ratios greater than 30 dB and tumor to fibroglandular ratios of 3 dB were measured. The encouraging images obtained with the system validate its potential as a clinical tool for breast cancer detection.

Abstract 1072: Development and characterization of patient derived xenograft (PDX) avatars of human cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is a fatal primary malignancy with limited treatment options and a dismal 5year survival rate of <10%. Poor prognosis and early recurrence after surgical treatment warrants further molecular characterization of these tumors to evaluate therapeutic sensitivities and identification of biomarkers for CCA. Although several Next Generation Sequencing (NGS) studies have revealed some targetable genomic aberrations, the lack of preclinical and translational models has severely limited the identification of clinically relevant targeted therapies for CCA. So far, very few putative molecular targets/pathways have been identified using patient-derived xenograft (PDX) models, CCA cell derived xenograft models (CDX) and CCA cell lines, only 4 of which have progressed to preclinical testing for therapeutic value. In this study we describe development and comprehensive characterization of patient derived xenograft models of CCA from freshly resected patient tissue. Patient tumor cells from 8 patients were engrafted subcutaneously in highly immunocompromised NSG (NOD.Cg-PrkdcscidIl2rgtm1Wj) mice for PDX development. Two (CCA-1 and CCA-7) of 8 (25 %) engraftments yielded successful PDX model. The PDX avatar and matched patient tumor samples were genomically characterized at the whole exome and transcriptome level to check the fidelity of PDX models. Exome sequencing analysis of PDX tumors demonstrate higher genomic aberrations as compared to their patient counterparts. Preliminary analysis of transcriptomic profiles from CCA TCGA dataset, long term established intra- and extra-hepatic CCA cell lines (Mz-ChA-1, TFK-1, SG231, CCLP-1, HuCC-T1 and HuH-28), CCA CDX model from Mz-ChA-1 cells and CCA PDX models demonstrate that CCA PDX models more faithfully recapitulate patient tumors as compared to long term established cell lines. In summary, this proof of concept study demonstrates that PDX avatars of CCA could serve as a good model system for preclinical target discovery and validation for a deadly and neglected disease such as CCA. Citation Format: Sanhita Rath, Sen Peng, Lauren Hartman, George Reid, Harshil Dhruv, Shannon Glaser, Fanyin Meng, Gianfranco Alpini, Scott Celinski. Development and characterization of patient derived xenograft (PDX) avatars of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1072.

Abstract 914: Development of a whole-urine, next-generation sequencing-based assay for early detection of aggressive prostate cancer

Abstract Despite advances in biomarker development, early detection of aggressive prostate cancer (PCa) remains challenging. Existing biomarkers show modest improvement over models based on serum prostate specific antigen (PSA). We have previously developed a clinical-grade laboratory-developed test, named MiProstate Score (MiPS), for individualized risk prediction of aggressive prostate cancer. It uses transcription-mediated amplification to quantify the gene-fusion TMPRSS2:ERG (T2:ERG) (T1E4 splice isoform) and the lncRNA PCA3 from whole-urine obtained after a digital rectal exam (DRE), combined with serum PSA. To improve MiPS, we describe here the pre-clinical development and validation of a targeted next generation sequencing assay (NGS-MiPS) using post-DRE urine RNA to asses ~90 PCa transcriptomic biomarkers. These include those in MiPS as well as many isoforms of common PCa gene fusions, mRNA, and lncRNA candidate biomarkers nominated by our large-scale PCa tissue RNAseq and other sources. We have obtained a 98% informative sample rate from 2.5 mL of urine and high technical reproducibility (Pearson r=0.99). Risk scores for having PCa [or high-grade PCa (Gleason Score >6)] on biopsy, as determined by clinical MiPS vs. the clinical MiPS model using NGS data, were highly concordant, Pearson’s r=0.74 (and r=0.81). Urine from patients with benign or Gleason 6 vs. Gleason ≥ 4+3=7 cancer on biopsy (extreme design) showed expected differences in the levels of T2:ERG T1E4 (p=0.00003) and PCA3 (p=0.07), with additional T2:ERG splice isoforms and other biomarkers also being significantly different between low vs. high grade cancer. Feature selection and logistic regression trained in an extreme design cohort (n=73) yielded a 29-transcript model that outperformed MiPS and serum PSA in two validation cohorts: 1. A held-out set from the extreme design cohort n=36, AUC 0.81 vs. 0.76 and 0.63 respectively; 2. A separate active surveillance cohort n=45, AUCs 0.66 vs. 0.56 and 0.53 respectively. These results support the potential utility of our urine based targeted NGS assay to supplement serum PSA for the early detection of aggressive prostate cancer. Citation Format: Andi K. Cani, Kevin Hu, Javed Siddiqui, Sumin Han, Daniel H. Hovelson, Chia-Jen Liu, Simpa S. Salami, Ganesh S. Palapattu, Todd M. Morgan, John T. Wei, Arul M. Chinnaiyan, Scott A. Tomlins. Development of a whole-urine, next-generation sequencing-based assay for early detection of aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 914.

Abstract 1941: Fluorescence lifetime imaging of treatment response in neuroendocrine tumor organoids

Abstract Gastroenteropancreatic neuroendocrine tumors (GEP-NET) are now the second most common form of gastrointestinal cancer, and account for roughly 60% of all neuroendocrine tumors. Unfortunately, no animal models or cell lines exist that reflect the in vivo characteristics of human GEP-NET’s for use as predictive models. Therefore, pre-clinical work is often not representative of the clinical outcome, so clinical trials begin in patients without rigorous pre-clinical validation of the treatment regimen. There is an established need to predict the most effective treatments prior to clinical trials for GEP-NET patients, to increase treatment efficacy and reduce toxicities. Here we show that a three-dimensional organoid model of primary patient tumors is representative of the original patient tumors. Furthermore, this study aims to use the organoid model to test a novel pre-clinical combination of ABT263, an experimental cancer drug that has been shown to enhance the effects of other chemotherapeutic agents, and the standard drug everolimus, on GEP-NET organoids. Previous studies have shown that ABT263, which targets Bcl-2, Bcl-XL and Bcl-w, induces apoptosis when used in combination with everolimus, making this a promising drug combination for GEP-NET patients. Treatment response to ABT263, everolimus, and the combination, was tested in three-dimensional organoids derived from six primary GEP-NET patients, using two-photon fluorescence lifetime imaging microscopy (FLIM). FLIM captures the fluorescence lifetime of metabolic cofactors NAD(P)H and FAD. Further, the fluorescence intensity of NAD(P)H divided by that of FAD (the redox ratio) was measured in all samples. Altogether, two-photon imaging of NAD(P)H and FAD fluorescence intensities and lifetimes (optical metabolic imaging, or OMI) in primary patient GEP-NET organoids provides a novel tool to measure metabolic changes with drug treatment. OMI measured metabolic differences induced by treatment in primary patient GEP-NETs when compared to control, at 72 hours post-treatment. All patients except one showed a decrease in redox ratio, which corresponds with a response, to the combination treatment. As metabolic imaging is performed to collect treatment response from more patients, this OMI data will be integrated into a multivariate analysis of variance to predict treatment response. Independent measures of organoid response, including changes in organoid diameter and immunohistochemistry, have been used to validate OMI predictions. We have shown that ABT263 plus everolimus is a promising combination treatment for GEP-NET’s. Overall, this work establishes the feasibility of personalized drug screens for GEP-NET’s using OMI in primary tumor organoids. Additionally, this platform can be used to identify new treatment regimens for improved GEP-NET patient outcomes, such as the ABT263 plus everolimus combination tested here. Citation Format: Amani A. Gillette, Christopher P. Babiarz, Cheri A. Pasch, Dustin A. Deming, Melissa C. Skala. Fluorescence lifetime imaging of treatment response in neuroendocrine tumor organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1941.

Abstract 4694: Evaluation of tumor-associated antigen expression with the MACSimaTM high-content imaging platform

Abstract Here we report the use of the MACSima™ imaging platform to perform high-content imaging for the pre-clinical validation of tumor target expression on tumor and healthy human tissues indicative for potential on-target/off-tumor toxicity in vivo. Major advances have been achieved in cancer therapy in the past decade. In particular, targeted-immunotherapy has progressed and clinical benefits for patients have been achieved. However, on-target/off-tumor toxicity is a potential threat which has been shown to be more pronounced in solid than in liquid tumors. These findings highlight the need for better pre-clinical assays to improve the safety profile of immunotherapies. On-target/off-tumor toxicity is mainly based on the expression of tumor-associated antigens (TAA) in healthy tissues under physiological conditions. Currently, most prediction methods for on-target/off-tumor expression are based on bulk mRNA expression data of healthy tissue. These prediction models, however, have limitations, mainly poor predictable relation between RNA and protein level. Moreover, it is frequently unclear which cell types are affected by on-target/off-tumor effects. To overcome these limitations we employ multi-parameter imaging to analyze the expression of TAAs directly at the protein level. Additionally, we gain spatial information about tissue and cellular distribution of TAAs. Notably, our novel high-content imaging technology potentially allows for the analysis of hundreds of markers in a single experiment, paving the way for high-dimensional characterization of cells within complex solid tissues. We performed high-content imaging with the MACSima™ platform to validate the expression of known TAAs across tumor and healthy tissue samples. Subsequently, we employed unbiased cluster analysis revealing correlation patterns within the datasets to identify cell types at risk. In detail, we analyzed several high-grade serous ovarian carcinoma and pancreatic ductal adenocarcinoma for the expression of known TAAs, described tumor markers, and tissue lineage markers. Additionally, we evaluated the expression of these TAAs across several healthy human tissues. Next we performed pixel- and object-based data analysis for unbiased cluster analysis. Thereby we identified cell clusters that express TAAs in ovarian and pancreatic cancers, as well as in healthy tissues. We found that primarily epithelial cells express the analyzed TAAs in different tissues and that TAA expression shows inter- and intra-tumor heterogeneity. Taken together, we established a novel workflow for multi-parameter characterization of tissues employing the MACSima™ imaging platform. Our work enables efficient identification of potential tissues for on-target/off-tumor toxicity, paving the way for improved pre-clinical evaluation of TAAs as new targets. Citation Format: Christoph Herbel, Vera Dittmer, Manuel Martinez-Osuna, Laura Nadine Kuester, Daniel Schaefer, Jan Drewes, Jutta Kollet, Werner Mueller, Michael Mallmann, Peter Mallmann, Philipp Stroebel, Olaf Hardt, Dominik Eckardt, Andreas Bosio. Evaluation of tumor-associated antigen expression with the MACSimaTM high-content imaging platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4694.

Abstract 4546: Functional cell surface proteomics of acute myeloid leukemia enables predictive modeling of antibody-drug conjugate cytotoxicity

Abstract Acute myeloid leukemia (AML) remains a significant unmet medical need. The development of targeted therapies for AML is challenging due to the heterogeneity of immunological and genetic disease subtypes and physiological similarities shared by blasts and normal myeloid progenitor cells. The identification of abundant cell surface markers on AML blasts with limited expression on bone marrow progenitors could drive the development of novel antibody-drug conjugates (ADCs) and other antibody-based therapeutics with improved therapeutic windows. Here we used hydrazide chemistry and mass spectrometry-based proteomics to profile the cell surface landscape of 95 primary blast samples from the Beat AML research consortium and normal bone marrow progenitor cells from healthy donors. We measured the abundance of over 5,000 proteins with high reproducibility (R2 = 0.89) and coverage of known cell surface markers (240/371). We also identified more than 2,500 N-glycosylation sites, many of which are predicted by canonical sequence motifs but have not been demonstrated experimentally. These findings thereby expand our knowledge of both the AML and normal hematological cell surface proteome. Additionally, we used pulsed stable isotope labeling coupled with the aforementioned hydrazide method to assess global surface protein abundance and turnover dynamics in a panel of malignant cell lines. We integrated our cell surface characterization with Beat AML’s mRNA expression, mutation, and AML subtype classification data to prioritize proteins for further exploration as candidate targets for ADC therapeutics. We generated a library of ADCs using monoclonal antibodies conjugated with Auristatin T, a highly potent membrane impermeable microtubule inhibitor, and measured ADC cytotoxic activity on a panel of AML cell lines as well as primary bone marrow progenitor cells. We used these integrated datasets to more precisely refine AML subtypes and identify patient populations that could benefit from novel AML ADCs. We report the preclinical validation of CD317 as a proof-of-concept for the utility of cell surface proteomics to identify novel targets for ADC therapeutics. Citation Format: Robert Lawrence, Robert Thurman, Travis Biechele, Cristina Tognon, Samantha Savage, Anna Reister Schultz, Jeffrey Tyner, William Arthur. Functional cell surface proteomics of acute myeloid leukemia enables predictive modeling of antibody-drug conjugate cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4546.

Abstract 3650: Integrative mass spectrometry and RNA-sequencing identifies DLK1 as a candidate immunotherapeutic target in neuroblastoma

Abstract BACKGROUND. Neuroblastoma (NB) is an embryonal tumor of the sympathetic nervous system that accounts for 12% of childhood cancer deaths. Despite multimodal therapy, survival probability for high-risk NB patients remains below 50% and relapsed NB is largely incurable. To date, the cell surface landscape (surfaceome) of NB remains poorly defined. An unbiased survey of these proteins will facilitate the identification of candidate immunotherapeutic targets for preclinical validation. METHODS. To identify proteins on the cell surface of NB cells, we performed plasma membrane protein extraction utilizing a sucrose density gradient methodology followed by nano-liquid chromatography coupled to mass spectrometry (nLC-MS/MS) in NB cell lines (n=12) and patient derived xenografts (PDX; n=10). We next integrated MS data with RNA-sequencing (NB=153; Normal=7859) data to evaluate proteins with an annotated extracellular domain differentially expressed in NB compared to normal tissues. Candidate immunotherapeutic targets were validated by immunohistochemistry on NB tumor and normal tissue microarray (TMA) and in-vitrofunctional studies were performed following genetic manipulation of candidate targets to assess cell proliferation, differentiation and viability. RESULTS. We yielded on average 66% (range:60-68%) membrane protein enrichment with high reproducibility between biological replicates (80%; range:78-84%) and identified 4826 unique membrane proteins. Our approach confirmed known cell surface proteins in development as immunotherapeutic targets in NB (ALK, GPC2, NCAM1, DLL3 and CD276). We prioritized DLK1 for further evaluation due to it being the only candidate with expression directly associated with a super enhancer element (P=6.09X10-5). Genetic depletion of DLK1 resulted in neurite outgrowth suggesting induction of terminal differentiation (P=7.26X10-5). Full proteome analysis of DLK1 knockdown and control using MS showed regulation of proteins that promote outgrowth of neurites (P=3.37X10-3) and development of neurons (P=3.76X10-3). CONCLUSION. We have developed the first MS-based surfaceome of NB. DLK1 is an epigenetically regulated oncoprotein in a large subset of high-risk NBs. We are currently developing antibody drug conjugate therapeutics designed for NBs with proven overexpression of DLK1. Citation Format: Amber K. Weiner, Alexander B. Radaoui, Nathan M. Kendsersky, Jo Lynne Harenza-Rokita, Simone Sidoli, Karina L. Conkrite, Zalman Vaksman, Komal Rathi, Pichai Raman, Daniel Martinez, Tricia Bhatti, Matthew Tsang, Bruce Pawel, Benjamin A. Garcia, John M. Maris, Sharon J. Diskin. Integrative mass spectrometry and RNA-sequencing identifies DLK1 as a candidate immunotherapeutic target in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3650.

Abstract LB-197: NBE-002, an anthracycline-based immune-stimulatory antibody drug conjugate (iADC) targeting ROR1 for the treatment of triple-negative breast cancer

Abstract Introduction: We report on the development and preclinical validation of NBE-002, a next-generation antibody drug conjugate (ADC) for the treatment of triple-negative breast cancer (TNBC). NBE-002 consists of a humanized monoclonal antibody directed against the receptor tyrosine kinase ROR1, expressed on the surface of numerous solid cancers including TNBC, and is site-specifically conjugated to a derivative of the highly potent anthracycline PNU-159682. Results: Therapeutic efficacy of NBE-002 was evaluated in ROR1-low/-intermediate/-high patient-derived xenograft (PDX) models of TNBC, lung adenocarcinoma, ovarian carcinoma, and a variety of sarcomas. NBE-002 was found to display significant anti-tumor activity in each indication. The most pronounced effect was achieved in TNBC, where complete tumor regression was observed already at the lowest ADC dose tested (0.33 mg/kg), even in models expressing low levels of ROR1 (H-score ≤70). Since these PDX studies were performed in immune-compromised hosts, and anthracyclines are known inducers of immunogenic cell death, anti-tumor activity was also investigated in syngeneic tumor models in immune-competent hosts. Anti-tumor activity of PNU-ADCs involved activation of the immune system, as shown by evaluation of NBE-002 or a Trastuzumab-PNU conjugate (T-PNU) in ROR1- or HER2-positive syngeneic breast cancer models, respectively. Depletion of CD8 T cells severely reduced anti-tumor activity, demonstrating an important role for T cells in driving tumor regression. Furthermore, when tumor free animals were re-challenged with the same tumor, tumor growth was inhibited in the absence of any further ADC administration, indicating the development of an immunological memory. Notably, combination of ADC and checkpoint inhibition, such as α-PD1 or α-CTLA4, significantly enhanced tumor eradication following the treatment. Conclusion: Our results demonstrate that NBE-002 is a highly effective and promising targeted therapeutic for the treatment of ROR1 positive TNBC and potentially other solid tumor indications that warrants clinical development. Considering the pronounced immune-modulatory functions of the PNU payload, NBE-002 may be particularly well suited for combination therapy with immune checkpoint inhibitors. NBE-002 is currently undergoing GMP manufacturing and initiation of clinical studies is expected in mid-2020. Citation Format: Roger R. Beerli, Lorenz Waldmeier, Rémy Gébleux, Francesca Pretto, Ulf Grawunder. NBE-002, an anthracycline-based immune-stimulatory antibody drug conjugate (iADC) targeting ROR1 for the treatment of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-197.

Potent neutralizing antibodies elicited by dengue vaccine in rhesus macaque target diverse epitopes.

There is still no safe and effective vaccine against dengue virus infection. Epidemics of dengue virus infection are increasingly a threat to human health around the world. Antibodies generated in response to dengue infection have been shown to impact disease development and effectiveness of dengue vaccine. In this study, we investigated monoclonal antibody responses to an experimental dengue vaccine in rhesus macaques. Variable regions of both heavy chain (VH) and light chain (VL) were cloned from single antibody-secreting B cells. A total of 780 monoclonal antibodies (mAbs) composed of paired VH and VL were characterized. Results show that the vaccination induces mAbs with diverse germline sequences and a wide range of binding affinities. Six potent neutralizing mAbs were identified among 130 dengue envelope protein binders. Critical amino acids for each neutralizing antibody binding to the dengue envelope protein were identified by alanine scanning of mutant libraries. Diverse epitopes were identified, including epitopes on the lateral ridge of DIII, the I-III hinge, the bc loop adjacent to the fusion loop of DII, and the β-strands and loops of DI. Significantly, one of the neutralizing mAbs has a previously unknown epitope in DII at the interface of the envelope and membrane protein and is capable of neutralizing all four dengue serotypes. Taken together, the results of this study not only provide preclinical validation for the tested experimental vaccine, but also shed light on a potential application of the rhesus macaque model for better dengue vaccine evaluation and design of vaccines and immunization strategies.

Establishment of a model of sentinel lymph node metastasis using immunodeficient swine

Lymph node metastasis occurs via the migration of cancer cells through the lymphatic system. Sentinel lymph node (SLN) biopsy is a common diagnostic strategy. SLNs have been studied using healthy rodents and large animals without metastasis. Here we used immunodeficient swine to establish a model of lymph node metastasis. We used RAG2-knockout immunodeficient swine. A431 human epithelial carcinoma cells expressing green fluorescent protein were injected subcutaneously into the posterior sides of the auricle, forelimb and hindlimb of knockout swine. Indigo carmine dye was injected subcutaneously 8 weeks after tumour cell transplantation. SLNs were extracted, observed using a stereoscopic fluorescence microscope and analysed histologically using haematoxylin and eosin staining, and immunohistochemistry. Lymphoid follicles were found in wild-type swine, and a few aggregated lymphocytes and immature lymphoid follicles were observed in knockout swine. Fluorescence in the lymph nodes indicated metastasis of tumour cells to the lymph nodes. Tumour cells replaced lymph node architectures, showed high-grade nuclear atypia and formed irregular tumour nests. Our model may be useful for the preclinical validation of diagnostic methods and minimally invasive treatment of metastatic cancer.

In a Model of Neuroinflammation Designed to Mimic Delirium, Quetiapine Reduces Cortisol Secretion and Preserves Reversal Learning in the Attentional Set Shifting Task

Quetiapine, an atypical antipsychotic medication has lacked pre-clinical validation for its purported benefits in the treatment of delirium. This laboratory investigation examined the effects of quetiapine on the attentional set shifting task (ASST), a measure of cognitive flexibility and executive functioning, in a rodent model of lipopolysaccharide (LPS) mediated neuroinflammation. 19 Sprague Dawley female rats were randomly selected to receive intraperitoneal placebo (N = 5), LPS and placebo (N = 7) or LPS and quetiapine (n = 7) and performed the ASST. We measured trials to criterion, errors, non-locomotion episodes and latency to criterion, serum cortisol and tumor necrosis factor alpha (TNF-α) levels. TNF-α levels were not different between groups at 24h. Cortisol levels in the LPS + Quetiapine group were reduced compared to LPS + Placebo (P < 0.001) and did not differ from the placebo group (P = 0.15). Analysis between LPS + Quetiapine and LPS + Placebo treated rats demonstrated improvement in the compound discrimination reversal (CD Rev1) (P = 0.016) and the intra-dimensional reversal (ID Rev2) (P = 0.007) discriminations on trials to criterion. LPS + Quetiapine treated rats had fewer errors than LPS + Placebo treated animals in the compound discrimination (CD) (P = 0.007), CD Rev1 (P = 0.005), ID Rev2 (P < 0.001) discriminations. There was no difference in non-locomotion frequency or latency to criterion between the three groups in all discriminations (P > 0.0167). We demonstrated preserved reversal learning, no effect on attentional set shifting and normalized cortisol levels in quetiapine-treated rats in this neuroinflammatory model of delirium. This suggests that quetiapine's beneficial effects in delirium may be related to the preservation of reversal learning and potential downstream effects related to reduction in cortisol production. Graphical Abstract.

Tropifexor-Mediated Abrogation of Steatohepatitis and Fibrosis Is Associated With the Antioxidative Gene Expression Profile in Rodents.

Farnesoid X receptor (FXR) agonism is emerging as an important potential therapeutic mechanism of action for multiple chronic liver diseases. The bile acid‐derived FXR agonist obeticholic acid (OCA) has shown promise in a phase 2 study in patients with nonalcoholic steatohepatitis (NASH). Here, we report efficacy of the novel nonbile acid FXR agonist tropifexor (LJN452) in two distinct preclinical models of NASH. The efficacy of tropifexor at <1 mg/kg doses was superior to that of OCA at 25 mg/kg in the liver in both NASH models. In a chemical and dietary model of NASH (Stelic animal model [STAM]), tropifexor reversed established fibrosis and reduced the nonalcoholic fatty liver disease activity score and hepatic triglycerides. In an insulin‐resistant obese NASH model (amylin liver NASH model [AMLN]), tropifexor markedly reduced steatohepatitis, fibrosis, and profibrogenic gene expression. Transcriptome analysis of livers from AMLN mice revealed 461 differentially expressed genes following tropifexor treatment that included a combination of signatures associated with reduction of oxidative stress, fibrogenesis, and inflammation. Conclusion: Based on preclinical validation in animal models, tropifexor is a promising investigational therapy that is currently under phase 2 development for NASH.

In vitro treatment of congenital disorder of glycosylation type Ia using PLGA nanoparticles loaded with GDP‑Man.

Congenital disorder of glycosylation (CDG) type Ia is a multisystem disorder that occurs due to mutations in the phosphomannomutase 2 (PMM2) gene, which encodes for an enzyme involved in the N‑glycosylation pathway. Mutated PMM2 leads to the reduced conversion of mannose‑6‑P to mannose‑1‑P, which results in low concentration levels of guanosine 5'‑diphospho‑D‑mannose (GDP‑Man), a nucleotide‑activated sugar essential for the construction of protein oligosaccharide chains. In the present study, an in vitro therapeutic approach was used, based on GDP‑Man‑loaded poly (D,L‑lactide‑co‑glycolide) (PLGA) nanoparticles (NPs), which were used to treat CDG‑Ia fibroblast cultures, thus bypassing the glycosylation pathway reaction catalysed by PMM2. To assess the degree of hypoglycosylation in vitro, the present study examined the activities of α‑mannosidase, β‑glucoronidase and β‑galactosidase in defective and normal fibroblasts. GDP‑Man (30 µg/ml GDP‑Man PLGA NPs) was incubated for 48 h with the cells and the specific activities of α‑mannosidase and β‑galactosidase were estimated at 69 and 92% compared with healthy controls. The residual activity of β‑glucoronidase increased from 6.5 to 32.5% and was significantly higher compared with that noted in the untreated CDG‑Ia fibroblasts. The glycosylation process of fibroblasts was also analysed by two‑dimensional electrophoresis. The results demonstrated that treatment caused the reappearance of several glycosylated proteins. The data in vitro showed that GDP‑Man PLGA NPs have desirable efficacy and warrant further evaluation in a preclinical validation animal model.

Preclinical validation of 3-phosphoinositide-dependent protein kinase 1 inhibition in pancreatic cancer

BackgroundThe very aggressive nature and low survival rate of pancreatic ductal adenocarcinoma (PDAC) dictates the necessity to find novel efficacious therapies. Recent evidence suggests that phosphoinositide 3-kinase (PI3K) and 3-phosphoinositide-dependent protein kinase 1 (PDK1) are key effectors of oncogenic KRAS in PDAC. Herein, we report the role and mechanism of action of PDK1, a protein kinase of the AGC family, in PDAC.MethodsPDAC cell lines were treated with selective PDK1 inhibitors or transfected with specific PDK1-targeting siRNAs. In vitro and in vivo assays were performed to investigate the functional role of PDK1 in PDAC. Specifically, anchorage-dependent and anchorage-independent growth was assessed in PDAC cells upon inhibition or downregulation of PDK1. Detailed investigation of the effect of PDK1 inhibition/downregulation on specific signalling pathways was also performed by Western blotting analysis. A xenograft tumour mouse model was used to determine the effect of pharmacological inhibition of PDK1 on PDAC cells growth in vivo.ResultsTreatment with specific inhibitors of PDK1 impaired anchorage-dependent and anchorage-independent growth of pancreatic cancer cell lines, as well as pancreatic tumour growth in a xenograft model. Mechanistically, inhibition or downregulation of PDK1 resulted in reduced activation of the serum/glucocorticoid regulated kinase family member 3 and subsequent reduced phosphorylation of its target N-Myc downstream regulated 1. Additionally, we found that combination of sub-optimal concentrations of inhibitors selective for PDK1 and the class IB PI3K isoform p110γ inhibits pancreatic cancer cell growth and colonies formation more potently than each single treatment.ConclusionsOur data indicate that PDK1 is a suitable target for therapeutic intervention in PDAC and support the clinical development of PDK1 inhibitors for PDAC.