Use of thermolytic protective groups to prevent G-tetrad formation in CpG ODN type D: structural studies and immunomodulatory activity in primates.
CpG oligodeoxynucleotides (ODN) show promise as immunoprotective agents and vaccine adjuvants. CpG ODN type D were shown to improve clinical outcome in rhesus macaques challenged with Leishmania major. These ODN have a self-complementary core sequence and a 3' end poly(G) track that favors G-tetrad formation leading to multimerization. Although multimerization appears necessary for localization to early endosomes and signaling via Toll-like receptor 9 (TLR-9), it can result in product polymorphisms, aggregation and precipitation, thereby hampering their clinical applications. This study shows that functionalizing the poly(G) track of D ODN with thermolytic 2-(N-formyl-N-methyl)aminoethyl (fma) phosphate/thiophosphate protecting groups (pro-D ODN) reduces G-tetrad formation in solution, while allowing tetrad formation inside the cell where the potassium concentration is higher. Temperature-dependent cleavage of the fma groups over time further promoted formation of stable G-tetrads. Peripheral blood cells internalized pro-D ODN efficiently, inducing high levels of IFNalpha, IL-6, IFNgamma and IP-10 and triggering dendritic cell maturation. Administration of pro-D35 to macaques challenged with L.major significantly increased the number of antigen-specific IFNgamma-secreting PBMC and reduced the severity of the skin lesions demonstrating immunoprotective activity of pro-D ODN in vivo. This technology fosters the development of more efficient immunotherapeutic oligonucleotide formulations for the treatment of allergies, cancer and infectious diseases.
Puig
Montserrat
M
Laboratory of Immunology, Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA.
Grajkowski
Andrzej
A
Boczkowska
Malgorzata
M
Ausín
Cristina
C
Beaucage
Serge L
SL
Verthelyi
Daniela
D
D000276:Adjuvants, Immunologic / Q000138:chemical synthesis / Q000737:chemistry* / Q000627:therapeutic use; D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D003470:Culture Media; D004247:DNA / Q000737:chemistry; D054856:G-Quadruplexes; D006147:Guanine / Q000737:chemistry; D006801:Humans; D016773:Leishmaniasis, Cutaneous / Q000188:drug therapy / Q000276:immunology; D008253:Macaca mulatta; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000627:therapeutic use; D011355:Prodrugs / Q000138:chemical synthesis / Q000737:chemistry*; D013696:Temperature
D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D003470:Culture Media; D009838:Oligodeoxyribonucleotides; D011355:Prodrugs; D006147:Guanine; D004247:DNA