title: Production and characterization of amplified tumor-derived cRNA libraries to be used as vaccines against metastatic melanomas.
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 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: CureVac, Paul Ehrlich Strasse 15, 72076 Tübingen, Germany. carralot@curevac.de
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Carralot
authors.forename: Benjamin
authors.initials: B
authors.lastname: Weide
authors.forename: Oliver
authors.initials: O
authors.lastname: Schoor
authors.forename: Jochen
authors.initials: J
authors.lastname: Probst
authors.forename: Birgit
authors.initials: B
authors.lastname: Scheel
authors.forename: Regina
authors.initials: R
authors.lastname: Teufel
authors.forename: Ingmar
authors.initials: I
authors.lastname: Hoerr
authors.forename: Claus
authors.initials: C
authors.lastname: Garbe
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Rammensee
authors.forename: Steve
authors.initials: S
authors.lastname: Pascolo