title: Enhancement of the Immunostimulatory Activity of a TLR7 Ligand by Conjugation to Polysaccharides.
abstract: Toll-like receptors (TLRs) in the innate immune system recognize specific pathogen-associated molecular patterns derived from microbes. Synthetic small molecule TLR7 agonists have been extensively evaluated as topical agents for antiviral and anticancer therapy, and as adjuvants for vaccine. However, safe and reproducible administration of synthetic TLR7 ligands has been difficult to achieve due to undesirable pharmacokinetics and unacceptable side effects. Here, we conjugated a versatile low molecular weight TLR7 ligand to various polysaccharides in order to improve its water solubility, enhance its potency, and maintain low toxicity. The synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine, designated 1V209, was stably conjugated to primary amine functionalized Ficoll or dextran using benzoic acid functional groups. The conjugation ratios using specified equivalents of TLR7 ligand were dose responsive and reproducible. The zeta potential value of the polysaccharides was decreased in inverse proportion to the ratio of conjugated TLR7 ligand. These conjugates were highly water-soluble, stable for at least 6 months at room temperature in aqueous solution, and easy to lyophilize and reconstitute without altering potency. In vitro studies with murine mononuclear leukocytes showed that the TLR7 agonist conjugated to polysaccharides had 10- to 1000-fold higher potencies than the unconjugated TLR7 ligand. In vivo pharmacodynamics studies after injection indicate that the conjugates induced systemic cytokine production. When the conjugates were used as vaccine adjuvants, they enhanced antigen specific humoral and cellular immune responses to a much greater extent than did unconjugated TLR7 ligands. These results indicated that small molecule TLR7 ligands conjugated to polysaccharides have improved immunostimulatory potency and pharmacodynamics. Polysaccharides can be conjugated to a variety of molecules such as antigens, peptides, and TLR ligands. Therefore, such conjugates could represent a versatile platform for the development of vaccines against cancer and infectious diseases.
authors.affiliation: †Moores Cancer Center, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, United States.|‡Department of Chemistry, Biotechnology and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Kohrimoto, Kagoshima 890-0065, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Shinchi
authors.affiliation: †Moores Cancer Center, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Crain
authors.affiliation: †Moores Cancer Center, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, United States.
authors.forename: Shiyin
authors.initials: S
authors.lastname: Yao
authors.affiliation: †Moores Cancer Center, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Chan
authors.affiliation: †Moores Cancer Center, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, United States.
authors.forename: Shannon S
authors.initials: SS
authors.lastname: Zhang
authors.affiliation: †Moores Cancer Center, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, United States.
authors.forename: Alast
authors.initials: A
authors.lastname: Ahmadiiveli
authors.affiliation: ‡Department of Chemistry, Biotechnology and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Kohrimoto, Kagoshima 890-0065, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Suda
authors.affiliation: †Moores Cancer Center, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, United States.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hayashi
authors.affiliation: †Moores Cancer Center, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, United States.
authors.forename: Howard B
authors.initials: HB
authors.lastname: Cottam
authors.affiliation: †Moores Cancer Center, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, United States.
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Carson
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage* / Q000737:chemistry*; D000818:Animals; D001853:Bone Marrow / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D005260:Female; D006801:Humans; D007114:Immunization; D007249:Inflammation / Q000188:drug therapy / Q000276:immunology* / Q000473:pathology; D008024:Ligands; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011134:Polysaccharides / Q000737:chemistry*; D051199:Toll-Like Receptor 7 / Q000502:physiology*
chemical_list: D000276:Adjuvants, Immunologic; D016207:Cytokines; D008024:Ligands; D011134:Polysaccharides; D051199:Toll-Like Receptor 7