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Castle J.C.,Merck And Co. | Castle J.C.,Institute for Translational Oncology and Immunology TRON | Biery M.,Merck And Co. | Biery M.,NuGEN Technologies | And 14 more authors.
BMC Genomics

Background: DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number.Results: We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual.Conclusion: The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads. © 2010 Castle et al; licensee BioMed Central Ltd. Source

Schnatbaum K.,JPT Peptide Technologies Gmbh | Schmoldt H.-U.,BioNTech AG | Daneschdar M.,BioNTech AG | Plum L.M.,Institute for Translational Oncology and Immunology TRON | And 9 more authors.
Biotechnology Journal

As membrane proteins play an important role in a variety of life-threatening diseases, the development of therapeutic monoclonal antibodies against membrane proteins is of significant interest. Among many other requirements, the process of antibody drug development requires a set of tailor-made assays for the characterization of the antibodies and for monitoring their activity. Designing assays to characterize antibodies directed to membrane proteins is challenging, because the natural targets are often not available in a format that is compatible with a biochemical assay setup. Thus, alternatives that mimic the targeted membrane proteins are needed. In this study, we developed optimal peptidic mimotopes for the ELISA-based detection of the novel therapeutic antibody IMAB362 in biological samples. Initial hits were identified using phage display and these hits were optimized with the help of structure-activity relationship analysis on peptide microarrays. The optimized peptides showed binding constants in the low nanomolar to picomolar range, an improvement by a factor of up to 30 compared to the initial hits. The best mimotope (apparent KD = 0.15 nM) was successfully used for the ELISA-based quantification of IMAB362 in samples from a mouse pharmacokinetic study. The process described allows the rapid discovery of mimotopes for target proteins that are difficult to produce or handle, which can then be used in pre-clinical and clinical assays or for the purification of biological products. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Kreiter S.,Institute for Translational Oncology and Immunology TRON | Diken M.,Institute for Translational Oncology and Immunology TRON | Selmi A.,Institute for Translational Oncology and Immunology TRON | Selmi A.,Johannes Gutenberg University Mainz | And 3 more authors.
Current Opinion in Immunology

While the endeavor to vaccinate against cancer has been pursued for over 20 years, only recently was the first tumor vaccine approved. Among the different antigen formats assessed for vaccination, coding messenger RNA (mRNA) is emerging as a particularly attractive option. It can code for all types of transcript based proteins, is easy and cost efficient to produce, has a favorable safety profile and enables induction of combined immune responses. Within the last few years major developments have been achieved in this field. Clinical approaches use mRNA either for direct administration or for engineering of adoptively transferred dendritic cells. However, there are still challenges to be overcome for successful clinical application of mRNA-based immunotherapies. © 2011 Elsevier Ltd. Source

Warminski M.,University of Warsaw | Kowalska J.,University of Warsaw | Buck J.,Ribological GmbH | Zuberek J.,University of Warsaw | And 8 more authors.
Bioorganic and Medicinal Chemistry Letters

Synthetic mRNA cap analogs are valuable tools in the preparation of modified mRNA transcripts with improved translational activity and increased cellular stability, and have recently attracted more attention because of their great potential in therapeutic applications. We have synthesized and tested isopropylidene dinucleotide cap analogs bearing a phosphorothioate group at the β position of the 5′,5′-triphosphate bridge (two diastereomers of 2′,3′-iPr-m7GppSpG), as synthetically simpler alternatives to previously obtained phosphorothioate cap analogs. To evaluate the utility of the new compounds in biological systems we determined their affinity to translation initiation factor 4E (eIF4E), and tested their translational properties in rabbit reticulocyte lysates (RRL) and in human immature dendritic cells (hiDCs). In order to explain the properties of isopropylidene analogs we performed 1H NMR conformational analysis and correlated the absolute configuration at the β-phosphorous atom with previously synthesized m7GppSpG. © 2013 Elsevier Ltd. All rights reserved. Source

Diken M.,Institute for Translational Oncology and Immunology TRON | Kreiter S.,Institute for Translational Oncology and Immunology TRON | Selmi A.,Institute for Translational Oncology and Immunology TRON | Britten C.M.,Johannes Gutenberg University Mainz | And 5 more authors.
Gene Therapy

Even though it is known for more than one decade that antigen-encoding RNA can deliver antigenic information to induce antigen-specific immunity against cancer, the nature and mechanism of RNA uptake have remained enigmatic. In this study, we investigated the pharmacokinetics of naked RNA administered into the lymph node. We observed that RNA is rapidly and selectively uptaken by lymph node dendritic cells (DCs). Furthermore, in vitro and in vivo studies revealed that the efficient internalization of RNA by human and murine DCs is primarily driven by macropinocytosis. Selective inhibition of macropinocytosis by compounds or as a consequence of DC maturation abrogated RNA internalization and delivery of encoded antigens. Our findings imply that bioavailability of recombinant RNA vaccines in vivo highly depends on the density and the maturation stage of DCs at the administration site and are of importance for the design of RNA-based clinical immunotherapy protocols. © 2011 Macmillan Publishers Limited All rights reserved. Source

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