Immunostimulatory PyNTTTTGT Oligodeoxynucleotides: Structural Properties and Refinement of the Active Motif

Abstract

It is well known that synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides within a given context stimulate B lymphocytes and plasmacytoid dendritic cells (PDCs) of the vertebrate immune system. We have reported that B lymphocyte and PDC stimulation in humans could also be efficiently achieved by using non-CpG ODNs bearing the immunostimulatory sequence (motif) PyNTTTTGT, wherein Py is C or T and N is any deoxyribonucleotide. We are now reporting a series of studies that gives further precision regarding the composition of this immunostimulatory motif. The analysis of hundreds of ODNs led us to the conclusion that the motif for optimal CpG-independent immune stimulation can be represented by a sequence of the following general formula: PyN(T/A)(T/C/G)(T/C/G)(T/G)GT, wherein Py is C or T and N is any deoxyribonucleotide and wherein at least two of the positions represented within parentheses are Ts. Requirements for optimal ODN activity are as follows: (1) at least one of the versions of the general motif must be located near the central portion of the ODN; and (2) the ODN must be 20 or more nucleotides long. PyN(T/A)(T/C/G)(T/C/G)(T/G)GT ODNs are active in a phosphorothioate or in a phosphodiester backbone. In a phosphodiester backbone a canonical motif is strongly required whereas in a phosphorothioate backbone specificity is, within certain limits, less strict. On the other hand, PyN(T/A)(T/C/G)(T/C/G)(T/G)GT oligonucleotides are inactive as a double chain or as a modified (phosphorothioate or hydroxyl-methyl modified) or unmodified RNA backbone.

Proposed mechanisms for oligonucleotide IMT504 induced diabetes reversion in a mouse model of immunodependent diabetes

“…2A), and IMT504 treatment, or saline as control, was commenced (day 1). Blood glucose increased progressively in STZ-saline mice during 10 wk of evaluation, whereas it decreased significantly in STZ-IMT504 mice (repeated-measures 2-way ANOVA, interaction P Ͻ 0.001, STZ-IMT504 different from STZ-saline on days 6,11,21,26,29,32,36,41,46,53, and 66, P Ͻ 0.01 or less; Fig. 2A).…”

“…of doses : 20). Glycemia was measured in tail blood samples in nonfasted conditions on days 1,6,11,17,21,26,29,32,36,41,46,53, and 66 at 11-12 AM (last IMT504 injection on day 40). Intraperitoneal glucose tolerance tests (IPGTT) were performed on day 60.…”

“…Our group has identified a class of non-CpG ODNs called PyNTTTTGT, characterized for their active site (Py is C or T, and N is A, T, C, or G). IMT504, the prototype of this class, has two specific PyNTTTTGT sequences in its molecule (41). CpG and PyNTTTTGT ODNs have a number of common characteristics but also remarkable differences (41).…”

“…IMT504, the prototype of this class, has two specific PyNTTTTGT sequences in its molecule (41). CpG and PyNTTTTGT ODNs have a number of common characteristics but also remarkable differences (41). Among the latter: 1) PyNTTTTGT ODNs do not induce IFN␣ secretion (13), 2) they stimulate granulocyte-macrophage colonystimulating factor (GM-CSF) secretion in human CD56ϩ (NK/ NKT) cells (42), and 3) they stimulate in vivo and in vitro MSCs proliferation (22).…”

Non-Clinical Safety Studies of IMT504, a Unique Non-CpG Oligonucleotide

“…These effects include in vitro activation, proliferation, and expression of costimulatory molecules on B cells (Elías et al, 2003;Rodriguez et al, 2006a); immune modulatory effect on mice, rats, and primates when combined with a variety of antigens Montaner et al, 2011Montaner et al, , 2012Gan et al, 2009) Statistically significant differences as compared with control (0 mg/kg/day). *p < 0.05; **p < 0.02. improvement of tissue damage or disease in animal models (Rodriguez et al, 2006b;Hernando-Insúa et al, 2007;Coronel et al, 2008;Bianchi et al, 2010).…”