Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists

doi:10.1016/j.addr.2008.12.008

Advanced Drug Delivery Reviews

Volume 61, Issue 3, 28 March 2009, Pages 195-204

https://doi.org/10.1016/j.addr.2008.12.008 Get rights and content

Abstract

Toll-like receptor 9 (TLR9) agonists have demonstrated substantial potential as vaccine adjuvants, and as mono- or combination therapies for the treatment of cancer and infectious and allergic diseases. Commonly referred to as CpG oligodeoxynucleotides (ODN), TLR9 agonists directly induce the activation and maturation of plasmacytoid dendritic cells and enhance differentiation of B cells into antibody-secreting plasma cells. Preclinical and early clinical data support the use of TLR9 agonists as vaccine adjuvants, where they can enhance both the humoral and cellular responses to diverse antigens. In mouse tumor models TLR9 agonists have shown activity not only as monotherapy, but also in combination with multiple other therapies including vaccines, antibodies, cellular therapies, other immunotherapies, antiangiogenic agents, radiotherapy, cryotherapy, and some chemotherapies. Phase I and II clinical trials have indicated that these agents have antitumor activity as single agents and enhance the development of antitumor T-cell responses when used as therapeutic vaccine adjuvants. CpG ODN have shown benefit in multiple rodent and primate models of asthma and other allergic diseases, with encouraging results in some early human clinical trials. Although their potential clinical contributions are enormous, the safety and efficacy of these TLR9 agonists in humans remain to be determined.

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".

¹: ●Top References.

View full text

Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists☆

Abstract

Introduction

Section snippets

TLR9 and the recognition of pathogen CpG DNA

Control mechanisms of CpG-mediated immune activation

Classes of synthetic CpG oligodeoxynucleotides

Development of CpG ODN as vaccine adjuvants

Approaches to the prevention and therapy of infectious disease using CpG ODN monotherapy

Anti-tumor therapy using CpG ODN

TLR9 and autoimmunity

Summary

Conflict of interest

Acknowledgement

Cell

J. Biol. Chem.

Immunity

Blood

Immunity

J. Invest. Dermatol.

Trends Immunol.

J. Biol. Chem.

Immunity

Curr. Biol.

J. Biol. Chem.

Blood

Cell

Bioorg. Med. Chem.

Vaccine

Vaccine

FEMS Immunol. Med. Microbiol.

FEMS Immunol. Med. Microbiol.

Exp. Gerontol.

Recognition of microorganisms and activation of the immune response

Nature

A Toll-like receptor recognizes bacterial DNA

Nature

CpG motifs in bacterial DNA trigger direct B-cell activation

Nature

CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: a double-blind phase I/II study

J. Clin. Immunol.

Potential role of phosphatidylinositol 3 kinase, rather than DNA-dependent protein kinase, in CpG DNA-induced immune activation

J. Exp. Med.

Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner

Eur. J. Immunol.

Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition

Proc. Natl. Acad. Sci. U. S. A.

TLR9 signals after translocating from the ER to CpG DNA in the lysosome

Nat. Immunol.

Toll-like receptor control of the adaptive immune responses

Nat. Immunol.

Human monocytes infected with Yersinia pestis express cell surface TLR9 and differentiate into dendritic cells

J. Immunol.

IFN-alpha regulates TLR-dependent gene expression of IFN-alpha, IFN-beta, IL-28, and IL-29

J. Immunol.

Human lung cancer cells express functionally active Toll-like receptor 9

Respir. Res.

Microbial DNA induces a host defense reaction of human respiratory epithelial cells

J. Immunol.

Expression of Toll-like receptor 9 and response to bacterial CpG oligodeoxynucleotides in human intestinal epithelium⁎

Clin. Exp. Immunol.

Spontaneous uptake of biologically active recombinant DNA by mammalian cells via a selected DNA segment

Gene Ther.

Uptake, cellular distribution and novel cellular binding proteins of immunostimulatory CpG oligodeoxynucleotides in glioblastoma cells

Mol. Cell. Biochem.

Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments

Eur. J. Immunol.

UNC93B1 delivers nucleotide-sensing Toll-like receptors to endolysosomes

Nature

A second binding site for double-stranded RNA in TLR3 and consequences for interferon activation

Nat. Struct. Mol. Biol.

Modulation of double-stranded RNA recognition by the N-terminal histidine-rich region of the human Toll-like receptor 3

J. Biol. Chem.

The molecular structure of the Toll-like receptor 3 ligand-binding domain

Proc. Natl. Acad. Sci. U.S.A.

Crystal structure of human Toll-like receptor 3 (TLR3) ectodomain

Science

Structural basis of Toll-like receptor 3 signaling with double-stranded RNA

Science

Ligand-induced conformational changes allosterically activate Toll-like receptor 9