Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells.
Adoptive transfer of dendritic cells (DCs) transfected with in vitro-transcribed, RNA-encoding, tumor-associated antigens has recently entered clinical testing as a promising approach for cancer immunotherapy. However, pharmacokinetic exploration of RNA as a potential drug compound and a key aspect of clinical development is still pending. While investigating the impact of different structural modifications of RNA molecules on the kinetics of the encoded protein in DCs, we identified components located 3' of the coding region that contributed to a higher transcript stability and translational efficiency. With the use of quantitative reverse transcription-polymerase chain reaction (RT-PCR) and eGFP variants to measure transcript amounts and protein yield, we showed that a poly(A) tail measuring 120 nucleotides compared with a shorter one, an unmasked poly(A) tail with a free 3' end rather than one extended with unrelated nucleotides, and 2 sequential beta-globin 3' untranslated regions cloned head to tail between the coding region and the poly(A) tail each independently enhanced RNA stability and translational efficiency. Consecutively, the density of antigen-specific peptide/MHC complexes on the transfected cells and their potency to stimulate and expand antigen-specific CD4+ and CD8+ T cells were also increased. In summary, our data provide a strategy for optimizing RNA-transfected DC vaccines and a basis for defining release criteria for such vaccine preparations.
Holtkamp
Silke
S
Division of Experimental and Translational Oncology, Department of Internal Medicine III, Johannes Gutenberg University, Obere Zahlbacherstr. 63, 55131 Mainz, Germany.
Kreiter
Sebastian
S
Selmi
Abderraouf
A
Simon
Petra
P
Koslowski
Michael
M
Huber
Christoph
C
Türeci
Ozlem
O
Sahin
Ugur
U
D000818:Animals; D017951:Antigen Presentation / Q000235:genetics / Q000276:immunology; D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D019496:Cancer Vaccines / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism* / Q000637:transplantation; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods; D008213:Lymphocyte Activation / Q000235:genetics / Q000276:immunology; D008297:Male; D051379:Mice; D009369:Neoplasms / Q000235:genetics / Q000276:immunology / Q000628:therapy; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability / Q000235:genetics / Q000276:immunology; D012334:RNA, Neoplasm / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D012334:RNA, Neoplasm; D011061:Poly A