pubs.acs.org/jmc Published on Web 08/03/2009 r 2009 American Chemical Society 5108 J. Med. Chem. 2009, 52, 5108–5114 DOI: 10.1021/jm900730r Modifications Incorporated in CpG Motifs of Oligodeoxynucleotides Lead to Antagonist Activity of Toll-like Receptors 7 and 9 Dong Yu, Daqing Wang, Fu-Gang Zhu, Lakshmi Bhagat, Meiru Dai, Ekambar R. Kandimalla, and Sudhir Agrawal* Idera Pharmaceuticals, Inc., 167 Sidney Street, Cambridge, Massachusetts 02139 Received May 28, 2009 Oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides act as agonists of TLR9 and induce Th1-type immune responses. In the present study, we synthesized CpG containing ODNs in which C or G was substituted with 2 0 -O-methylribonucleotides, 5-methyl-dC, or 2 0 -O-methyl-5-methyl- C and studied their immune stimulatory activity alone and in combination with TLR agonists. In mouse and human primary cell-based assays, modified ODNs did not stimulate immune responses but inhibited TLR9 agonist-induced immune stimulatory activity. In mice, modified ODNs did not induce cytokines but inhibited immune responses induced by agonists of TLR7 and TLR9. Modified ODNs did not inhibit endosomal TLR3- or cell-surface TLR4-agonist-induced cytokines. This study demonstrates that ODNs incorporated with chemical modifications in CpG dinucleotides do not induce immune stimulatory activity but act as antagonists of TLR7 and TLR9 in vitro and in vivo. These types of modifications are commonly employed in antisense sequences and thereby may affect the intended mechanism of action. Introduction Toll-like receptors (TLRs a ) are a family of transmembrane proteins that detect pathogen-associated molecular patterns and elicit pathogen-specific immune responses. 1 Of the 10 TLRs identified in humans, TLRs 3, 7, 8, and 9 are expressed in endosomal membranes and recognize pathogen-derived nucleic acid molecular patterns. 1 Synthetic, bacterial, and viral DNA containing unmethylated CpG dinucleotides act as ligands of TLR9 and are widely studied. 2-5 We have extensively studied the structure-activity relation- ships of ODNs containing CpG motifs and identified certain nucleotide, backbone, and linker modifications that, upon site-specific incorporation in the flanking sequence 5 0 or 3 0 to the CpG dinucleotide, have significant influence on immune stimulatory activity. 6-13 Previously we reported that incorpora- tion of 2 0 -O-methylribonucleotides in ODNs in the first or second nucleotide position adjacent to the CpG dinucleotide on the 5 0 -side abrogates immune stimulatory activity. 7,12,13 In recent studies, we observed that presence of 2 0 -O-methylribo- nuclotide substitutions in flanking sequences to CpG motif not only neutralize immune stimulatory activity but also act as antagonists of TLR7 and TLR9 in vitro and in vivo in mice. 14 Recent studies have shown that TLR9 exists in dimer form and the binding of CpG ODN to the receptor causes con- formational changes in the cytoplasmic signaling domain of the receptor, thus leading to the recruitment of adapter molecules and activation of immune signaling pathways. 15 ODNs lacking a CpG motif also bind to TLR9 but are not capable of bringing conformational changes in the signaling domain, thus failing to activate immune stimulation. 15 Our previous studies showed that substitution of either C or G of the CpG dinucleotide with 2 0 -O-methyl-C or -G, respectively, or C with 5-methyl-dC or a methylphosphonate linkage between C and G resulted in a significant decrease in immune stimulatory activity. 16 Such modifications are commonly employed in antisense ODNs containing CpG dinucleotides to minimize TLR9-mediated immune responses. On the basis of our most recent results, 14 we hypothesized that ODNs containing these substitutions in the CpG motif do not show TLR9-mediated immune responses because they do not bring conformational changes in the signaling domain of TLR9 but may act as antagonists of TLR9. In the present study, we synthesized ODNs containing a CpG dinucleotide with 5-methyl-dC, 2 0 -O-methyl-C, or 2 0 -O- methyl-5-methyl-C substituted for C and/or 2 0 -O-methyl-G substituted for G and studied their immune stimulatory properties including antagonist activity. These studies were carried out in various cell-based assays including TLR9 transfected HEK293 cells, mouse spleen cell cultures, and in vivo in mice. We observed that these ODNs containing modified CpG dinucleotides are not immune stimulatory but act as antagonists of TLR7 and TLR9. Materials and Methods Synthesis and Purification of Oligodeoxynucleotides. All ODNs and their controls shown in Table 1 were synthesized, purified, and analyzed as previously reported. 14 All ODNs were characterized by capillary gel electrophoresis (CGE) or denaturing polyacrylamide gel electrophoresis (PAGE) and *To whom correspondence should be addressed. Phone: 617-679- 5501. Fax: 617-679-5572. E-mail: sagrawal@iderapharma.com. a Abbreviations: CGE, capillary gel electrophoresis; CpG, deoxycy- tidine-phosphate-deoxyguanosine; ELISA, enzyme-linked immuno- sorbent assay; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; IFN, interferon; NF-κB, nuclear factor κB; ODN, oligodeoxynucleotide; PBMCs, peripheral blood mononuclear cells; PAGE, polyacrylamide gel electrophoresis; TLR, Toll-like receptor.