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.

Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7

“…For example, the adenine derivative 1V270 was conjugated to a phospholipid via its N 9 on the purine ring (46). Via the same site, another adenine derivative 1V209 was attached to polysaccharides (47). The same nitrogen, numbered N 1 in tricyclic derivatives of the -quimod series (numbered I or 1 in Figure S1 in Supplementary Material), was used for conjugation of an imiquimod derivative to nanogels (48).…”

Differential Role of B Cells and IL-17 Versus IFN-γ During Early and Late Rejection of Pig Islet Xenografts in Mice

“…Several possible pathways may be triggered by carbohydrate ligands, leading to enhanced IL-17 production. For example, carbohydrate ligands may conjugate with endogenous TLR ligands and enhance their immunostimulatory activity on TLRs(41, 42), leading to enhanced inflammation conducive for IL-17 production. Alternatively, carbohydrate may directly interact with lectin receptors and license lectin-bearing APCs to differentiate effector cells towards IL-17 production(43–45).…”

Delivery of Immunotherapeutic Nanoparticles to Tumors via Enzyme‐Directed Assembly

“…However, despite their promise, immunotherapeutics face significant challenges. First, many powerful immunostimulatory molecules suffer from solubility and stability issues that prevent their direct translation as clinical therapeutics . Second, off‐target delivery of these compounds can cause nonspecific immune activation and toxicity .…”

“…Despite promising results, the application of the small molecule 1V209 alone is hampered by poor solubility and excessive cytokine induction upon systemic administration. These drawbacks limit the working concentration of this compound …”

“…This orthotopic syngeneic mouse model of breast cancer was chosen as it is highly metastatic, known to overexpress MMPs, and has been shown to respond to immunotherapy . Sera were collected at 2 h following IV injection of each formulation and the levels of proinflammatory cytokines IP‐10, MCP‐1, and IL‐6 in plasma were determined (Figure A–C), as these proinflammatory cytokines have been shown to be upregulated at 2 h following systemic administration of 1V209 . No significant upregulation of these cytokines was observed in animals administered with either 1V209‐ l ‐NP and 1V209‐ d ‐NP .…”