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Production and characterization of amplified tumor-derived cRNA libraries to be used as vaccines against metastatic melanomas

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10 pages
Anti-tumor vaccines targeting the entire tumor antigen repertoire represent an attractive immunotherapeutic approach. In the context of a phase I/II clinical trial, we vaccinated metastatic melanoma patients with autologous amplified tumor mRNA. In order to provide the large quantities of mRNA needed for each patient, the Stratagene Creator™ SMART™ cDNA library construction method was modified and applied to produce libraries derived from the tumors of 15 patients. The quality of those mRNA library vaccines was evaluated through sequencing and microarray analysis. Results Random analysis of bacterial clones of the library showed a rate of 95% of recombinant plasmids among which a minimum of 51% of the clones contained a full-Open Reading Frame. In addition, despite a biased amplification toward small abundant transcripts compared to large rare fragments, we could document a relatively conserved gene expression profile between the total RNA of the tumor of origin and the corresponding in vitro transcribed complementary RNA (cRNA). Finally, listing the 30 most abundant transcripts of patient MEL02's library, a large number of tumor associated antigens (TAAs) either patient specific or shared by several melanomas were found. Conclusion Our results show that unlimited amounts of cRNA representing tumor's transcriptome could be obtained and that this cRNA was a reliable source of a large variety of tumor antigens.
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Genetic Vaccines and Therapy
BioMedCentral
Open Access Research Production and characterization of amplified tumor-derived cRNA libraries to be used as vaccines against metastatic melanomas 1,2 32 1,2 JeanPhilippe Carralot, BenjaminWeide ,Oliver Schoor, JochenProbst , 1 11 3 Birgit Scheel, Regina Teufel, Ingmar Hoerr, Claus Garbe, Hans 2 1,2 Georg Rammenseeand Steve Pascolo*
1 2 Address: CureVac,Paul Ehrlich Strasse 15, 72076 Tübingen, Germany,University of Tübingen, Institute for Cell Biology, Department of 3 Immunology; Auf der Morgenstelle 15; 72076 Tübingen, Germany andSection for Dermatological Oncology, Tübingen University Hospital, Liebermeisterstraße 25, 72076 Tübingen, Germany Email: JeanPhilippe Carralot  carralot@curevac.de; Benjamin Weide  bnweide@med.unituebingen.de; Oliver Schoor  oliver.schoor@uni tuebingen.de; Jochen Probst  jochen.probst@student.unituebingen.de; Birgit Scheel  bs@curevac.de; Regina Teufel  rt@curevac.de; Ingmar Hoerr  ih@curevac.de; Claus Garbe  claus.garbe@med.unituebingen.de; HansGeorg Rammensee  rammensee@unituebingen.de; Steve Pascolo*  steve.pascolo@unituebingen.de * Corresponding author
Published: 22 August 2005Received: 22 June 2005 Accepted: 22 August 2005 Genetic Vaccines and Therapy2005,3:6 doi:10.1186/1479-0556-3-6 This article is available from: http://www.gvt-journal.com/content/3/1/6 © 2005 Carralot et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Background:Anti-tumor vaccines targeting the entire tumor antigen repertoire represent an attractive immunotherapeutic approach. In the context of a phase I/II clinical trial, we vaccinated metastatic melanoma patients with autologous amplified tumor mRNA. In order to provide the large quantities of mRNA needed for each patient, the Stratagene Creator™ SMART™ cDNA library construction method was modified and applied to produce libraries derived from the tumors of 15 patients. The quality of those mRNA library vaccines was evaluated through sequencing and microarray analysis. Results:Random analysis of bacterial clones of the library showed a rate of 95% of recombinant plasmids among which a minimum of 51% of the clones contained a full-Open Reading Frame. In addition, despite a biased amplification toward small abundant transcripts compared to large rare fragments, we could document a relatively conserved gene expression profile between the total RNA of the tumor of origin and the correspondingin vitrotranscribed complementary RNA (cRNA). Finally, listing the 30 most abundant transcripts of patient MEL02's library, a large number of tumor associated antigens (TAAs) either patient specific or shared by several melanomas were found. Conclusion:Our results show that unlimited amounts of cRNA representing tumor's transcriptome could be obtained and that this cRNA was a reliable source of a large variety of tumor antigens.
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