ISS have received considerable attention due to their potential for treatment of a wide range of disorders, including asthma, allergy, infectious diseases, and cancer (reviewed in 33
). The optimal recognition motif for human cells includes a CG dinucleotide preceded by a T (16
). We recently extended this finding by demonstrating that the minimal human immunostimulatory CpG motif for the potent induction of IFN-α and IFN-γ from human cells is 5′-TCGXX (22
). In order to extend the motif and multimeric presentation studies with short ISS, we designed and tested a series of linear and branched CICs containing heptameric ISS connected with a variety of non-nucleoside spacers all linked through phosphorothioate groups. Similarly to microparticle-formulated short ISS, CICs with a heptameric sequence containing a 5′-TCG gave statistically significant production of IFN-α and IFN-γ from human PBMC relative to the non-CpG controls. Interestingly, while short ISS/microparticle complexes showed no preference for the bases following the 5′-TCG in either IFN-α or IFN-γ induction, CICs containing a 5′-TCG plus a second CpG within the heptameric sequence (5′-TCGXCGX, 5′-TCGXTCG) induced higher levels of IFN-α/IFN-γ than those with only a 5′-TCG (5′-TCGXXXX).
The nature of the non-nucleoside spacers and the overall structure of the CIC substantially influenced the induction of IFN-α, but not IFN-γ, in human PBMC cultures. For branched CICs built on a glycerol scaffold, increasing the length of the spacers between the ISS and the glycerol backbone correlated with augmented IFN-α production, with long, hydrophilic spacers based on HEG being the most potent spacers tested. Additionally, IFN-α production dramatically increased as more free 5′-TCG-containing heptameric ISS were added to the CIC. The Ficoll-based CIC containing 186 free 5′-TCGACGT sequences, C22, was the most active IFN-α-inducing CIC tested and exhibited a bell-shaped dose–response curve. This type of IFN-α dose–response curve was also observed for a new class of conventional ISS identified by our laboratory, called CpG-C, which are characterized by their ability to induce high levels of IFN-α from PBMC and also stimulate B cells (17
). It is important to note that despite the enhanced IFN-α-inducing properties of certain branched CICs, they continue to activate the same pattern of gene expression as 1018 ISS, indicating that they are transmitting similar, but enhanced, signals as conventional ISS.
Our laboratory has generated increasing amounts of data supporting the view that multimeric presentation of ISS significantly increases IFN-α induction from PBMC. Presentation of two or more copies of an active ISS via a soluble (glycerol, trebler or Ficoll for CICs) or insoluble (microparticles for heptameric ISS) support results in compounds that strongly induce IFN-α (22
). Additionally, members of a class of conventional ISS, called CpG-A, are also known to be high IFN-α inducers (16
) and form large aggregates due to a combination of their self-complimentary regions and their poly-guanosine motifs, which facilitate the formation of high-order secondary structures (17
). The specific function of multimeric presentation of active ISS motifs is not yet clear. It is quite possible that TLR9 cross-linking enhances or alters signal transduction, especially the pathways leading to IFN-α expression. Alternatively, the binding of ISS by TLR9 may be weak so that the increased avidity possible with multivalent molecules may be needed for optimum signaling.
Human B lymphocytes are TLR9-positive and directly responsive to ISS and CICs. Our data demonstrate that B cell activity is primarily dependent on the ISS sequence within the CIC, with 5′-TCGTXXX and 5′-AACGTTC exhibiting the most potent activity and 5′-TCGAXXX sequences being the least active. Contrary to findings regarding IFN-α induction, spacer composition and multimeric display of 5′-ISS via branched CICs exhibited less influence on B cell activation. These distinctly different patterns of ISS response suggest that a fundamental difference exists in the manner in which IFN-α-producing cells versus B cells take up and/or recognize ISS. Remarkably, CICs can be uniquely designed such that the B cell activity can vary from 10 to 100% of the activity of 1018 ISS, while maintaining high levels of IFN-α production, a feature that could be highly useful for the treatment of certain diseases that might benefit from an IFN-α-mediated response in the absence of polyclonal B cell stimulation (e.g. asthma).
It is of primary importance to screen and optimize ISS and CICs for activity specifically in human cells because of the differences in sequence motif requirements and TLR9+
cell types in human versus mouse (37
). Nevertheless, it can be convenient to introduce mouse activity into an optimized human ISS or CIC in order to simplify in vivo
evaluation in non-human systems. Thus, we prepared a series of CICs containing the optimal sequences, spacers and structures for IFN-α/IFN-γ induction from human cells, but replaced one of the human ISS sequences with a mouse-active sequence. These hybrid CICs maintained similar levels of IFN-α and IFN-γ induction as the fully human-active CICs, while also demonstrating good cytokine-inducing activity in mouse splenocytes. The in vitro
activity was confirmed by the in vivo
study in mice, which showed that CICs induce similar levels of ISS-responsive cytokines and chemokines in lung cells following an intranasal delivery.
Results from studies with longer ISS ODNs connected by spacers have been reported by Agrawal and colleagues (38
). Those studies differed from our work by using ISS ODNs containing only mouse-specific motifs and assaying for activity with mouse, not human, cells. The optimal spacers in that system were found to be short, alkyl spacers based on C3
) or C4
, rather than HEG (1
), which we found to be essential in CICs for high IFN-α induction from human cells. In more recent work, this group also demonstrated that short 5′-TCGTT-containing ISS of 5–7 bases in length linked with glycerol and additional C3
spacers induced cytokine production in mouse cultures and modest induction in a human PBMC sample, although IFN-α was not measured (39
). In contrast, our studies have concentrated on the activities of branched CICs, with which we demonstrated that in human cells, only IFN-α secretion and IFN-α-related gene expression are increased by compounds with multiple 5′-ends, while other ISS-inducible activities, such as IFN-γ induction, B lymphocyte proliferation and IL-6 production, are unaffected by multimeric delivery. Additionally, another element that appears to be crucial for high IFN-α induction by branched CICs is that the ISS motifs are presented via a long, hydrophilic spacer, such as HEG, possibly because less constrictive spacing allows the heptamers to avoid sterically hindering each other from each associating with a receptor. These findings underscore the importance of sequences, spacers, and structures for deriving optimal ISS activity in the human system.
In conclusion, by using CICs composed of short heptameric CpG DNA sequences linked with non-nucleoside spacers, we have identified optimal motifs for ISS-mediated IFN-α induction from human PBMC, human B cell activation and mouse activity and have demonstrated that some of these activities can be independently modulated. Significantly, identification of the optimal motifs, spacers and structures for these different biological properties allows for the assembly of CICs with a defined set of activities that can be tailored for particular applications. Our major efforts have been directed toward identification of CICs with higher IFN-α-inducing activity, as opposed to high B cell stimulation, because there is little evidence thus far that the clinically important activities of ISS are mediated by polyclonal B cell activation. In fact, for certain indications, including the treatment of allergic asthma and certain cancers, it would be desirable to avoid polyclonal B cell activation, which might result in the potentiation of asthma-mediating B cells or B cell lymphomas. As such, CICs may offer a unique advantage over conventional ISS in clinical applications that require a restricted set of immune-enhancing activities.