Synthesis and Characterization of PEGylated Toll Like Receptor 7 Ligands

Abstract

Toll like receptor 7 (TLR7) is located in the endosomal compartment of immune cells. Signaling through TLR7, mediated by the adaptor protein MyD88, stimulates the innate immune system and shapes adaptive immune responses. Previously, we characterized TLR7 ligands conjugated to protein, lipid or polyethylene glycol (PEG). Among the TLR7 ligand conjugates, the addition of PEG chains reduced the agonistic potency. PEGs are safe in humans and widely used for improvement of pharmacokinetics in existing biologics and some low molecular weight compounds. PEGylation could be a feasible method to alter the pharmacokinetics and pharmacodynamics of TLR7 ligands. In this study, we systematically studied the influence of PEG chain length on the in vitro and in vivo properties of potent TLR7 ligands. PEGylation increased solubility of the TLR7 ligands and modulated protein binding. Adding a 6-10 length PEG to the TLR7 ligand reduced its potency toward induction of interleukin (IL)-6 by murine macrophages in vitro and IL-6 and tumor necrosis factor (TNF) in vivo. However, PEGylation with 18 or longer chain restored, and even enhanced, the agonistic activity of the drug. In human peripheral blood mononuclear cells, similar effects of PEGylation were observed for secretion of proinflammatory cytokines, IL-6, IL-12, TNF-α, IL-1β and type 1 interferon, as well for B cell proliferation. In summary, these studies demonstrate that conjugation of PEG chains to a synthetic TLR ligand can impact its potency for cytokine induction depending on the size of the PEG moiety. Thus, PEGylation may be a feasible approach to regulate the pharmacological properties of TLR7 ligands.

Enhancement of the Immunostimulatory Activity of a TLR7 Ligand by Conjugation to Polysaccharides

“…6,11,14 Improvement of immunostimulatory potencies of small molecule TLR7 ligands have been achieved by conjugation and formulation. 17,18,41,42 In previous studies, we demonstrated that conjugation of macromolecules, such as albumin, phospholipids and polyethylene glycol, to small molecule TLR7 ligands enhanced the immunostimulatory activities and efficacy as vaccine adjuvants. 17,18 Kedl, et al also reported that the conjugation of TLR7 ligand to proteins is necessary for enhancement of immunostimulatory activity.…”

“…17,18,41,42 In previous studies, we demonstrated that conjugation of macromolecules, such as albumin, phospholipids and polyethylene glycol, to small molecule TLR7 ligands enhanced the immunostimulatory activities and efficacy as vaccine adjuvants. 17,18 Kedl, et al also reported that the conjugation of TLR7 ligand to proteins is necessary for enhancement of immunostimulatory activity. 41 Polysaccharides have been used to increase immunostimulatory potency of TLR9 ligands.…”

“…17,18 Imiquimod and resiquimod do not contain such a "handle" and the effects of modification of the magnitude we required here are unknown.…”

“…6,[13][14][15] In previous studies, we conjugated a synthetic small molecule purine-like TLR7 ligand to various hydrophilic macromolecules, including polyethylene glycol and serum albumin, to improve water solubility. 17,18 We demonstrated that conjugation to serum albumin increased the potency 10-to 100-fold in vitro and improved its pharmacodynamics in vivo. 17 However, serum albumin is a biological and may not be an ideal adjuvant due to its physical instability and potential microbial contamination.…”

“…Previous experiments indicated that conjugation of TLR7 ligands to macromolecules altered in vivo behavior of the TLR7 ligand. 18 To study the pharmacodynamics (PD) of the conjugates in vivo, C57BL/6 mice were intravenously injected with 100 nmol, based on the concentration of TLR7 ligand, of the 400 kDa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Immunization study of TLR7 ligand-polysaccharide conjugates with Ovalbumin as antigen.…”