Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists☆
Introduction
The innate immune system detects and responds to invading microbes and viruses using several highly-conserved families of receptors that can recognize pathogen-expressed molecules [1]. The most well understood of these receptors are the Toll-like receptors (TLRs), of which a family of 10 related molecules has been identified in humans. Some of the TLRs detect molecules that are highly specific for microbes, such as lipopolysaccharide, which is detected by TLR4. In contrast, TLR3, TLR7, and TLR8 detect RNA, and TLR9 detects DNA, that is in principle quite similar between viruses, bacteria, and vertebrates. The TLRs evolved to detect infections and to trigger protective innate and adaptive immune responses. In the absence of infection synthetic TLR ligands can be used to trigger selective immune responses that can provide therapeutic benefits in several disease settings. This chapter will focus on the therapeutic applications of stimulating TLR9, which is normally activated by unmethylated CpG dinucleotides [2]. These “CpG motifs” are relatively common in bacterial and viral DNA, but are suppressed and methylated in vertebrate DNA [3].
For therapeutic applications TLR9 is commonly activated by synthetic oligodeoxynucleotides (ODN) containing one or more CpG motifs [3]. To reduce nuclease degradation, therapeutic CpG ODN usually have at least partially phosphorothioate-modified backbones. The first human clinical trial of a CpG ODN began in 1999, and showed encouraging evidence of activity as a vaccine adjuvant [4].
Section snippets
TLR9 and the recognition of pathogen CpG DNA
At the time of the first report describing CpG-dependent immune stimulation by bacterial DNA, the receptor was not yet identified [3]. An early study indicated that the catalytic subunit of the DNA-dependent protein-kinase (DNA-PKcs), involved in the repair of DNA double-strand breaks, is the mediator of CpG innate immune activation [5], although these studies could not be confirmed [6]. Gene knock down and gain of function experiments finally identified TLR9 to be the receptor conferring CpG
Control mechanisms of CpG-mediated immune activation
Immune responses stimulated upon TLR9 engagement are tightly controlled. Specific control mechanisms evolved regulating excessive bias in TLR9 responses at different levels of TLR9 signaling and cytokine induction, adding to the complexity of TLR9-mediated immune activation. Cyclooxygenase-2 (COX-2), NO synthase 2 (NOS2), and production of nitric oxide, prostaglandin E2 (PGE2) or the regulatory cytokine IL-10 are up-regulated upon CpG activation and down-regulate CpG effects [46], [47], [48],
Classes of synthetic CpG oligodeoxynucleotides
The immune stimulatory effects of CpG DNA are explained at least in part by differences inherent to genomic DNA of vertebrates and pathogens: vertebrate CpG dinucleotides are methylated and their frequency is suppressed, while viral and bacterial CpG dinucleotides are non-methylated and occur with a much higher frequency [3]. Synthetic CpG ODN can be generated containing specific CpG sequence motifs, sugar, base or backbone modifications as well as secondary and tertiary structures that all
Development of CpG ODN as vaccine adjuvants
By stimulating innate immunity, TLR9 activation also enhances antigen-specific humoral and cellular immune responses. Nearly all of the vaccine studies performed to date have used B-Class CpG ODN, on which this section will focus. However, C-Class ODN also have shown vaccine adjuvant activity, and so the same general principles are expected to apply as well to these drug candidates [●75]. Mechanisms contributing to the strong adjuvant activity of these CpG ODN for inducing humoral immunity may
Approaches to the prevention and therapy of infectious disease using CpG ODN monotherapy
The Th1-like immune effects of TLR9 activation appear to have evolved to stimulate protective immunity against intracellular pathogens. Consistent with this role, studies from many investigators have demonstrated that prophylactic treatment of mice with a synthetic TLR9 ligand can provide transient protection against a wide range of viral, bacterial, and even some parasitic pathogens, including lethal challenge with some Category A agents or surrogates such as B. anthracis, vaccinia virus, F.
Anti-tumor therapy using CpG ODN
Although there has been great interest in the development of tumor vaccines, the level of success achieved to date using this type of approach has been disappointing [159]. As reviewed above, the early results with CpG ODN as vaccine adjuvants offer some hope that this approach may yet enjoy some greater clinical success. TLR9 activation with PF-3512676 or other CpG ODN has been shown to induce a Th1-like cytokine response in humans with B cell lymphomas, suggesting a potential role for this
TLR9 and autoimmunity
Vertebrate DNA is methylated and in contrast to DNA of a variety of bacterial and viral pathogens does normally not induce TLR9-mediated innate immune effects [3], [174], [175]. Although vertebrate DNA also contains DNA sequences that are suppressive to TLR9 activation [175], enforced endosomal translocation and prevention of DNA digestion by uptake enhancers or in the form of immune complexes result in TLR9-dependent signaling [64], [176], [177], [178]. Cell surface expression enables TLR9 to
Summary
The stimulation of the immune system by synthetic CpG ODN via TLR9 leads to a variety of directed effects linking innate to adaptive immune responses. TLR9 agonists can be used as highly effective targeted immune modulators with broad potential applications as vaccine adjuvants, and as stand-alone therapy or in combination with other therapies in cancer, infectious diseases or asthma and allergy. Animal models and ongoing phase I to phase III clinical trials suggest that CpG ODN have anti-tumor
Conflict of interest
Both authors are employees of Pfizer, are co-inventors on patents relating to TLR9 agonists, and have a financial interest in the therapeutic development of TLR9 agonists.
Acknowledgement
The authors thank Silke Fähndrich for expert assistance in manuscript preparation.
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This review is part of the Advanced Drug Delivery Reviews theme issue on "CpG Oligonucleotides as Immunotherapeutic Adjuvants: Innovative Applications and Delivery Strategies".
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