title: Conjugation of ligands at the 5'-end of CpG DNA affects immunostimulatory activity.
abstract: Bacterial DNA and synthetic oligonucleotides containing unmethylated CpG dinucleotides (CpG DNA) activate the vertebrate immune system and promote Th1-like immune responses. Several CpG DNAs are currently being tested in clinical trials as either alone or in combination with vaccines, antibodies, and allergens separately or as conjugates for a number of disease indications including cancers, allergies, and asthma. In this paper, we show that conjugation of an oligonucleotide and a CpG DNA through their 5'-ends (5'-5'-linked DNA) significantly reduces the immunostimulatory activity of the CpG DNA. In addition, we found that the reduction in immunostimulatory activity of 5'-5'-linked CpG DNA depends on the size of the oligonucleotide conjugated to CpG DNA. Conjugation of a smaller group or molecule, such as a phosphorothioate group, at the 5'-end of CpG DNA has an insignificant effect on immunostimulatory activity. However, conjugation of a mononucleotide, tetra- or longer oligonucleotide or a fluorescein molecule to the 5'-end of a CpG DNA (5'-5'-linked DNA) significantly suppresses the immunostimulatory activity of CpG DNA. Surprisingly, conjugation of an oligonucleotide or a ligand through the 3'-end of CpG DNA (3'-3'-linked DNA) has an insignificant effect on immunostimulatory activity. Studies of cellular uptake and activation of transcription factor NF-kappaB in J774 cells using fluorescein-conjugated CpG DNAs suggest that the differences in the immune stimulation of 5'- and 3'-end-conjugated CpG DNAs is not as a result of differences in their cellular uptake properties. These results suggest that for optimal immunostimulatory activity, ligands should not be attached at the 5'-end of the CpG DNA.
authors.affiliation: Hybridon, Inc., 345 Vassar Street, Cambridge, Massachusetts 02139, USA.
authors.forename: Ekambar R
authors.initials: ER
authors.lastname: Kandimalla
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Bhagat
authors.forename: Dong
authors.initials: D
authors.lastname: Yu
authors.forename: Yanping
authors.initials: Y
authors.lastname: Cong
authors.forename: Jimmy
authors.initials: J
authors.lastname: Tang
authors.forename: Sudhir
authors.initials: S
authors.lastname: Agrawal
mesh_terms: D024509:3' Flanking Region; D024506:5' Flanking Region; D000276:Adjuvants, Immunologic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents; D016207:Cytokines / Q000378:metabolism; D005260:Female; D007107:Immune System / Q000166:cytology / Q000187:drug effects*; D008024:Ligands; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000187:drug effects; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000276:immunology*; D009841:Oligonucleotides / Q000737:chemistry / Q000276:immunology; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D003432:Cross-Linking Reagents; D016207:Cytokines; D008024:Ligands; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides