Synthetic small molecule Toll like receptor 7 (TLR7) ligands are effective topical agents for anti-viral and anti-tumor therapy by activation of innate immune response in the host. However, currently available small molecule synthetic TLR7 ligands cannot be administrated systemically due to low solubility, rapid pharmacokinetics, and associated cytokine release syndrome. Conjugation of a TLR9 ligand to polysaccharides was shown to be safe and effective (Marshall, et al., Nucleic Acid Res., 2003). Here, we hypothesized that conjugation of a low molecular weight TLR7 ligand to polysaccharide would increase water solubility, and improve its PK. The synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine (1V209), was conjugated to 400 kDa Ficoll (3, 18, and 39 equivalent (eq) of 1V209/molecule), 70 kDa (1, 5, and 11 eq of 1V209/molecule), 500 kDa (5, 22, and 46 eq of 1V209/molecule), and 2,000 kDa (4, 18, and 54 eq of 1V209/molecule) dextran using amine functionalized groups on polysaccharides and benzoic acid functional groups on the TLR7 ligand. The conjugation ratios using specified equivalents of 1V209 were dose responsive and reproducible. The number of residual amines and zeta potential were decreased in inverse proportion to the ratio of conjugated 1V209. These conjugates were highly water soluble, stable for at least 4 weeks at room temperature in aqueous solution, and easy to lyophilize and reconstitute. In vitro studies with murine mononuclear leukocyte showed 10 to 100 times higher potencies of Ficoll- or dextran-conjugated TLR7 ligands compared to unconjugated drug, respectively. Interestingly, the molecular weights of the polysaccharides or the average number of TLR7 ligands per molecule did not significantly influence in vitro potencies correlating to concentration of 1V209. In vivo pharmacodynamics studies showed that the conjugates induced systemic cytokine production compared to unconjugated drugs. When the conjugates were used as a vaccine adjuvant, the Ficoll-1V209 conjugates enhanced antigen specific humoral and cellular immune responses to a greater extent compared to unconjugated TLR7 ligands. These data indicate that small molecule TLR7 ligands conjugated to polysaccharides improve immunostimulatory properties as well as the drug like properties of the ligands. We hypothesize that various polysaccharide antigens derived from cancers or microbes can be effectively conjugated to TLR7 ligands to create potent new vaccines.

Enhancement of potency of the TLR7 ligand by conjugation to polysaccharide

249th National Meeting (2015)

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