Therapeutic Implications of Orally Delivered Immunomodulatory Oligonucleotides
Authors: Dennis M Klinman
Journal: Molecular Therapy (2015)

DNA has multiple and complex effects on the immune system. Bacterial DNA contains immunostimulatory CpG motifs that trigger cells expressing Toll-like receptor 9 (TLR9) to proliferate, mature, and support T helper type 1 (Th1)-biased immune responses (reviewed in ref. 1Krieg AM Therapeutic potential of Toll-like receptor 9 activation.Nat Rev Drug Discov. 2006; 5: 471-484Crossref PubMed Scopus (1052) Google Scholar). This response confers a selective advantage to the host by improving resistance to infection. In contrast, mammalian DNA contains sequence motifs that downregulate the immune system. Synthetic oligodeoxynucleotides that mimic this inhibitory activity (iODNs) slow or prevent the development of inflammatory and autoimmune diseases, consistent with the release of DNA from dying host cells inhibiting autoaggressive immune responses (reviewed in ref. 2Klinman DM Tross D Klaschik S Shirota H Sato T Therapeutic applications and mechanisms underlying the activity of immunosuppressive oligonucleotides.Ann NY Acad Sci. 2009; 1175: 80-88Crossref PubMed Scopus (30) Google Scholar). As reported in this issue, Wang et al. break new ground in the study of immunomodulatory ODNs by showing that the severity of chemically induced atopic dermatitis can be reduced by oral delivery of iODNs and worsened by CpG ODNs.3Wang Y Yamamoto Y Shigemori S Watanabe T Oshiro K Wang X et al.Inhibitory/suppressive oligodeoxynucleotide nanocapsules as simple oral delivery devices for preventing atopic dermatitis in mice.Mol Ther. 2015; 23: 297-309Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Wang et al. encapsulated these ODNs (1% by weight) in carbonate–apatite nanoparticles designed to resist stomach acids that would otherwise degrade unencapsulated ODNs. Their nanoparticles, measuring 100–200 nm, were selectively adsorbed from the gut by macrophages present in the Peyer's patches. This differs from the pattern of ODN uptake following parenteral administration, in which lymphocytes, monocytes, and dendritic cells as well as macrophages internalize and respond to these agents.1Krieg AM Therapeutic potential of Toll-like receptor 9 activation.Nat Rev Drug Discov. 2006; 5: 471-484Crossref PubMed Scopus (1052) Google Scholar,4Klinman DM Immunotherapeutic uses of CpG oligodeoxynucleotides.Nat Rev Immunol. 2004; 4: 249-258Crossref PubMed Scopus (820) Google Scholar To examine whether orally ingested ODNs affect systemic immunity, the authors monitored lymphocyte function ex vivo. Spleen cells from mice fed encapsulated iODN nanoparticles (iODNcaps) plus allergen produced less interleukin-4 (IL-4) and IL-33 than controls. This reduction in systemic immunity was attributed to iODNcaps blocking STAT6 phosphorylation, which inhibited the differentiation of allergen-activated Th2 cells into IL-4 producers.5Ito Y Shigemori S Sato T Shimazu T Hatano K Otani H et al.Class I/II hybrid inhibitory oligodeoxynucleotide exerts Th1 and Th2 double immunosuppression.FEBS Open Bio. 2013; 3: 41-45Crossref PubMed Scopus (18) Google Scholar By contrast, spleen cells from mice fed CpGcaps produced more interferon-γ (IFN-γ) and IL-33 after re-stimulation, consistent with the amplification of Th1-biased immunity observed by other groups after parenteral CpG administration.1Krieg AM Therapeutic potential of Toll-like receptor 9 activation.Nat Rev Drug Discov. 2006; 5: 471-484Crossref PubMed Scopus (1052) Google Scholar,4Klinman DM Immunotherapeutic uses of CpG oligodeoxynucleotides.Nat Rev Immunol. 2004; 4: 249-258Crossref PubMed Scopus (820) Google Scholar The utility of CpG ODNs for cancer immunotherapy, as vaccine adjuvants, and for the treatment of infection and allergy has been examined in preclinical and/or phase I–III trials.6Scheiermann J Klinman DM Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer.Vaccine. 2014; 32: 6377-6389Crossref PubMed Scopus (230) Google Scholar Studies of iODNs (also referred to in the literature as suppressive ODNs) have generally been performed using murine models of various autoimmune and inflammatory diseases.2Klinman DM Tross D Klaschik S Shirota H Sato T Therapeutic applications and mechanisms underlying the activity of immunosuppressive oligonucleotides.Ann NY Acad Sci. 2009; 1175: 80-88Crossref PubMed Scopus (30) Google Scholar,5Ito Y Shigemori S Sato T Shimazu T Hatano K Otani H et al.Class I/II hybrid inhibitory oligodeoxynucleotide exerts Th1 and Th2 double immunosuppression.FEBS Open Bio. 2013; 3: 41-45Crossref PubMed Scopus (18) Google Scholar,7Lenert P Stunz L Yi AK Krieg AM Ashman RF CpG stimulation of primary mouse B cells is blocked by inhibitory oligodeoxyribonucleotides at a site proximal to NF-kappaB activation.Antisense Nucleic Acid Drug Dev. 2001; 11: 247-256Crossref PubMed Scopus (104) Google Scholar Results show that iODNs reduce susceptibility to Th2-dependent allergens and slow or prevent the progression of diseases characterized by excessive immune activation.2Klinman DM Tross D Klaschik S Shirota H Sato T Therapeutic applications and mechanisms underlying the activity of immunosuppressive oligonucleotides.Ann NY Acad Sci. 2009; 1175: 80-88Crossref PubMed Scopus (30) Google Scholar The mechanisms by which iODNs influence disease susceptibility are still under investigation but include blocking the phosphorylation of STATs 1, 4, and 6, thereby inhibiting the signal transduction cascade needed to maintain inflammatory and autoimmune conditions.5Ito Y Shigemori S Sato T Shimazu T Hatano K Otani H et al.Class I/II hybrid inhibitory oligodeoxynucleotide exerts Th1 and Th2 double immunosuppression.FEBS Open Bio. 2013; 3: 41-45Crossref PubMed Scopus (18) Google Scholar,8Shirota H Gursel I Gursel M Klinman DM Suppressive oligodeoxynucleotides protect mice from lethal endotoxic shock.J Immunol. 2005; 174: 4579-4583Crossref PubMed Scopus (82) Google Scholar The activity of several different classes of iODNs is determined by the inclusion of poly-G motifs, telomeric repeats, and/or ability to form secondary or tertiary structures.9Kamdar K Nguyen V DePaolo RW Toll-like receptor signaling and regulation of intestinal immunity.Virulence. 2013; 4: 207-212Crossref PubMed Scopus (61) Google Scholar The iODNs used by Wang et al.3Wang Y Yamamoto Y Shigemori S Watanabe T Oshiro K Wang X et al.Inhibitory/suppressive oligodeoxynucleotide nanocapsules as simple oral delivery devices for preventing atopic dermatitis in mice.Mol Ther. 2015; 23: 297-309Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar (5'-CCTCATTAGGGTGAGGG-3') contain G-rich regions that facilitate the formation of complex intrachain and interchain Hoogsteen hydrogen bonds.10Murchie AI Tetraplex folding of telomere sequences and the inclusion of adenine bases.EMBO J. 1994; 13: 993-1001PubMed Google Scholar,11Gursel I Gursel M Yamada H Ishii KJ Takeshita F Klinman DM Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation.J Immunol. 2003; 171: 1393-1400Crossref PubMed Scopus (206) Google Scholar These iODNs can downregulate the production of IL-6, IL-4, IL-12, and IFN-γ (9Kamdar K Nguyen V DePaolo RW Toll-like receptor signaling and regulation of intestinal immunity.Virulence. 2013; 4: 207-212Crossref PubMed Scopus (61) Google Scholar), an effect abrogated by the elimination of the telomeric repeats.11Gursel I Gursel M Yamada H Ishii KJ Takeshita F Klinman DM Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation.J Immunol. 2003; 171: 1393-1400Crossref PubMed Scopus (206) Google Scholar iODNs also reduce serum autoantibody levels in models of autoimmune disease consistent with iODNs inhibiting the production of cytokines, chemokines, and antibodies by self-reactive lymphocytes. Optimal control of inflammatory/autoimmune processes by iODNs requires that these agents be administered early in the disease process. Progressively less benefit accrues if iODN treatment is delayed. For example, iODNs prolong the survival of young but not aged lupus-prone mice and are effective in the treatment of toxic shock and silicosis only if administered concurrent with the induction of those diseases.2Klinman DM Tross D Klaschik S Shirota H Sato T Therapeutic applications and mechanisms underlying the activity of immunosuppressive oligonucleotides.Ann NY Acad Sci. 2009; 1175: 80-88Crossref PubMed Scopus (30) Google Scholar In the study by Wang et al.,3Wang Y Yamamoto Y Shigemori S Watanabe T Oshiro K Wang X et al.Inhibitory/suppressive oligodeoxynucleotide nanocapsules as simple oral delivery devices for preventing atopic dermatitis in mice.Mol Ther. 2015; 23: 297-309Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar dermatitis was induced by repeatedly applying the contact-sensitizing agent picryl chloride (PiCl) to the skin. Inflammation first appeared after the third weekly application of PiCl and then worsened over time. Treatment with iODNcaps was initiated concurrent with the first application of PiCl (before disease had been induced) and continued daily for the duration of the experiment. When evaluating allergic responses, CpGs and iODNs were delivered orally to mice starting one week before initial allergen exposure and continuing daily throughout the period of antigen administration. Thus, results from these studies support the use of iODNs to reduce host susceptibility to developing disease but do not demonstrate their usefulness in treating established illness (as would be present in patients seeking medical attention). Moreover, the apparent necessity for long-term administration of iODNs increases the possibility that undesirable side effects (such as reduced responsiveness to immunization or infectious challenge) may accrue. Several issues concerning the activity of orally delivered ODNcaps require further exploration. Wang et al. did not evaluate ODN pharmacokinetics after oral administration and thus did not elucidate what fraction of CpGs or iODNs was absorbed, how long they persisted, or how they were distributed in vivo. While macrophages from the Peyer's patches were found to ingest ODNcaps, no evidence was presented to show that uptake activated these macrophages in vivo or caused them to migrate to sites of dermatitis or produce factors capable of altering systemic immunity. To verify that ingested iODNs are well suited to the treatment of atopic dermatitis, a comparison between oral vs. parenteral therapy would be helpful. Indeed, as the amount of ODNs persisting in the gastrointestinal tract following oral delivery was not examined, it is possible that changes observed in systemic immunity were mediated by ODN-induced changes in gut immunity or the microbiome rather than Peyer's patch macrophages. Such possibilities are supported by recent reports that TLR agonists alter the microbiome and older literature showing that CpG ODNs are effective adjuvants when delivered orally.9Kamdar K Nguyen V DePaolo RW Toll-like receptor signaling and regulation of intestinal immunity.Virulence. 2013; 4: 207-212Crossref PubMed Scopus (61) Google Scholar,12McDermott AJ Huffnagle GB The microbiome and regulation of mucosal immunity.Immunology. 2014; 142: 24-31Crossref PubMed Scopus (194) Google Scholar,13McCluskie MJ Davis HL Oral, intrarectal and intranasal immunizations using CpG and non-CpG oligodeoxynucleotides as adjuvants.Vaccine. 2000; 19: 413-422Crossref PubMed Scopus (145) Google Scholar Physicians rarely recommend the use of agents that require long-term parenteral administration to patients who have yet to develop disease. However, the use of oral agents that reduce disease susceptibility is extremely common (including drugs that reduce the risk of heart disease, stroke, and diabetes). Previous clinical trials examining the therapeutic potential of immunomodulatory ODNs generally evaluated their activity after parenteral injection.6Scheiermann J Klinman DM Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer.Vaccine. 2014; 32: 6377-6389Crossref PubMed Scopus (230) Google Scholar In murine models of arthritis, iritis, and silicosis, iODNs were maximally active when delivered directly to the joints, eyes, and lungs, respectively.2Klinman DM Tross D Klaschik S Shirota H Sato T Therapeutic applications and mechanisms underlying the activity of immunosuppressive oligonucleotides.Ann NY Acad Sci. 2009; 1175: 80-88Crossref PubMed Scopus (30) Google Scholar Similarly, the anti- tumor activity of CpG ODNs was maximized by direct injection into the tumor bed.14Shirota Y Shirota H Klinman DM Intratumoral injection of CpG oligonucleotides induces the differentiation and reduces the immunosuppressive activity of myeloid-derived suppressor cells.J Immunol. 2012; 188: 1592-1599Crossref PubMed Scopus (184) Google Scholar Those findings supported the general perception that immunomodulatory ODNs should be administered parenterally, preferably to the site of disease. Yet Wang and colleagues’ results3Wang Y Yamamoto Y Shigemori S Watanabe T Oshiro K Wang X et al.Inhibitory/suppressive oligodeoxynucleotide nanocapsules as simple oral delivery devices for preventing atopic dermatitis in mice.Mol Ther. 2015; 23: 297-309Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar indicate that oral delivery of both stimulatory and inhibitory ODNs alter immune function and disease susceptibility systemically. This finding is of considerable importance, as it suggests that the therapeutic utility of these agents is far broader than previously imagined. If clinical efficacy can be achieved after oral ODN administration, the cost, complexity, and inconvenience of immunomodulatory therapy would be dramatically reduced. The assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the National Cancer Institute at large. Members of Dr. Klinman's lab have patents related to the use of suppressive oligonucleotides. All rights to such patents have been assigned to the federal government.