Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

13.4 Backbone-dependent Immune Effects of CpG Motifs and Delineation &
an extremely high degree of nuclease resistance and greatly stabilizes the ODN
against degradation [68]. The phosphorothioate ODN backbone dramatically increases
the non-specific ODN binding to a wide variety of proteins [69, 70]Ç Phosphorothioate
ODN bind much more avidly to cell membranes, and generally have a
much higher degree of cell uptake [63, 71, 72]Ç The phosphorothioate backbone results
in sequence-independent activities including the activation of SP1 transcription
factor activity [73], inhibition of smooth muscle cell proliferation and migration [74,
75]Ë inhibition of basic fibroblast growth factor binding to its receptor [76, 77] and angiogenic
activity [78], reduction of the sequence specific binding of transcription factors
to their binding sites [79], inhibition of cellular adhesion to extracellular matrix
[80], enhancement of LPS-induced TNF production [81], and some degree of non-sequence
specific immune stimulation [82]. The immune stimulatory effects of PS
ODN are reduced by further modification with 2' methoxyethoxy modifications [83].
Finally, the PS backbone enhances certain effects of poly(G) sequences, which may
form G-quartets, including the ability to inhibit CD28 expression and in vivo contact
hypersensitivity responses [84].
PS ODN are approximately 200 times more potent at activating B cells than the
same sequence with a PO backbone [32, 63, 85, 86]Ç However, the immune-stimulatory
effects of CpG motifs in PS ODN are qualitatively different from PO ODN. PS
ODN bearing suboptimal CpG motifs are less likely than PO ODN to drive B cell
proliferation, especially if the CpG is followed by a G. PS ODN without CG motifs
are also frequently observed to non-specifically drive the proliferation of murine and
human B cells, although to a more limited degree than that which occurs with CpG
ODN [87, 66, 82, 88]Ç PS non-CpG ODN can synergize with strong signals for B cell
stimulation [89]. In contrast to their powerful B cell-stimulating activity, PS ODN are
less active than ODN in which the CpG motif is PO when assayed for their ability to
activate macrophages or NK cells [90±92].
As a result of the effects reviewed above, ODN with different backbones and different
sequence motifs induce dramatically different profiles and kinetics of immune
activation [90, 93±99]. The term ªCpG DNAº should therefore be used with care,
since not all of the effects described in the literature are seen with all ODN, bacterial
genomic DNAs or plasmids containing CpG motifs. Throughout this chapter effort
will be made to clarify when the effects described are unique to one or another form
of CpG DNA.
ODN containing PO backbones, or in which the CpG motifs are PO, are referred to
as CpG-A ODN, based on their potent activity at inducing IFN-a production from
plasmacytoid DC precursors and activating NK cells [86, 90, 98, 100±102]. The NK
cell activation and induction of plasmacytoid DC IFN-a expression that is induced by
an optimal CpG-A ODN is far higher than that induced by bDNA and the level of B
cell activation is generally lower. Even in comparing bDNA from different bacteria,
there are striking differences in the levels of immune activation; E. coli DNA tending
to be much more stimulatory than that from C. perfringens, a kind of bacteria or
Streptococcal or Staphylococcal sp. [8, 9, 39, 103]. ODN in which the 5' and 3' ends are
phosphorothioate-modified and the center portion is phosphodiester, have a high degree
of nuclease resistance provided by the phosphorothioate-modified ends, and
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