Agonists of Toll-like receptors (TLRs) are potent activators of the innate immune system and hold promise as vaccine adjuvant and for anticancer immunotherapy. Unfortunately, in soluble form they readily enter systemic circulation and cause systemic inflammatory toxicity. Here we demonstrate that by covalent ligation of a smallmolecule imidazoquinoline-based TLR7/8 agonist to 50-nm-sized degradable polymeric nanogels the potency of the agonist to activate TLR7/8 in in vitro cultured dendritic cells is largely retained. Importantly, imidazoquinoline-ligated nanogels focused the in vivo immune activation on the draining lymph nodes while dramatically reducing systemic inflammation. Mechanistic studies revealed a prevalent passive diffusion of the nanogels to the draining lymph node. Moreover, immunization studies in mice have shown that relative to soluble TLR7/8 agonist, imidazoquinoline-ligated nanogels induce superior antibody and T-cell responses against a tuberculosis antigen. This approach opens possibilities to enhance the therapeutic benefit of small-molecule TLR agonist for a variety of applications.nanotechnology | Toll-like receptor | dendritic cells | lymph node | vaccine A ctivation of the innate immune system crucially depends on the recognition of evolutionary conserved microbe-associated molecular patterns by host pattern recognition receptors (PRRs). Triggering of PRRs not only elicits a direct antimicrobial and inflammatory cascade but also activates the necessary antigen-presenting cells to subsequently prime antigen-specific T-and B-cell immune responses (1). Agonists of Toll-like receptors (TLRs) are among the most potent activators of the innate immune response identified to date and, thus, are under intensive investigation as adjuvants for vaccination (2) or as agents for anticancer immunotherapy (3). Especially promising in this context are agonists of the endosomal TLR7 and TLR8, which typically recognize singlestranded RNAs generated during viral infection (4) but can also be activated by synthetic small-molecule agonists (5). Molecular adjuvants (6, 7) that are agonists of TLR7/8 indeed activate a broad spectrum of antigen-presenting cells, both in mice (only TLR7) and humans, and typically induce high levels of type I IFN and IL-12, the most vital cytokines to drive the Th1 and cytotoxic T-cell responses required to combat intracellular infections and cancer (7,8).Due to their pharmacokinetic profile, most molecular adjuvants rapidly diffuse after administration and evoke systemic inflammatory responses that cause dose-limiting toxicity (9, 10). In the context of intratumoral administration, these dose-limiting toxicities prevent these compounds from reaching the necessary intratumoral concentration to yield real therapeutic benefit. In the context of vaccination, the rapid diffusion of molecular adjuvants dramatically lowers the ability of antigen and TLR agonist to reach the same antigen-presenting cells in the draining lymph node (DLN), which results in suboptimal immunity to the del...

MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anti-cancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface displayed L-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis we synthesized a 20-amino acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An L-Rha-cholesterol conjugate was prepared using tetraethylene glycol (TEG) as a linker. The liposome-based anti-cancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the non-immunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells. Because this vaccine uses separate rhamnose and antigenic epitope components, the vaccine can easily be targeted to different antigens or epitopes by changing the peptide without having to change the other components.

Toll-like receptor (TLR) 7 and 8 agonists are potential vaccine adjuvants, since they directly activate APCs and enhance Th1-driven immune responses. Previous SAR investigations in several scaffolds of small molecule TLR7/8 activators pointed to the strict dependence of the selectivity for TLR7 vis-à-vis TLR8 on the electronic configurations of the heterocyclic systems, which we sought to examine quantitatively with the goal of developing “heuristics” to define structural requisites governing activity at TLR7 and/or TLR8. We undertook a scaffold-hopping approach, entailing the syntheses and biological evaluations of 13 different chemotypes. Crystal structures of TLR8 in complex with the two most active compounds confirmed important binding interactions playing a key role in ligand occupancy and biological activity. Density functional theory based quantum chemical calculations on these compounds followed by linear discriminant analyses permitted the classification of inactive, TLR8-active, and TLR7/8 dual-active compounds, confirming the critical role of partial charges in determining biological activity.

Human Toll-like receptor 8 (hTLR8) is expressed in myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells. Engagement by TLR8 agonists evokes a distinct cytokine profile which favors the development of type 1 helper T cells. Crystal structures of the ectodomain of hTLR8 cocrystallized with two regioisomers of a dual TLR7/8-agonistic N1-substituted imidazoquinolines showed subtle differences in their interactions in the binding site of hTLR8. We hypothesized that the potency of a previously reported best-in-class pure TLR8 agonist, 3-pentylquinoline-2-amine, could be further enhanced by “designing in” functional groups that would mimic key intermolecular interactions that we had observed in the crystal structures. We performed a focused exploration of decorating the quinoline core with alkylamino groups at all possible positions. These studies have led to the identification of a novel TLR8 agonist that was ∼20-fold more potent than the parent compound and displays prominent adjuvantic activity in a rabbit model of immunization.

Activation of human toll-like receptor-8 (TLR8), expressed in myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells, evokes a distinct cytokine profile which favors the development of Type 1 helper T cells. Part-structures of the 2-aminobenzimidazole scaffold were examined with a view to identifying structural requisites corresponding to the smallest possible fragment of the benzimidazole core that would allow for retention of TLR8-agonistic activity. TLR8-specific agonistic activity was retained in 1-pentyl-4-phenyl-1H-imidazol-2-amine. The crystal structure of this compound bound to the TLR8 ectodomain displayed binding interactions that are common to other TLR8 agonists. This compound showed markedly attenuated proinflammatory properties in ex vivo human blood models. SAR studies revealed that 4-(2-(benzyloxy)phenyl)-1-pentyl-1H-imidazol-2-amine inhibited TLR signaling in a variety of TLR reporter cell lines, as well as in pharmacologically relevant human blood model systems. A kinase screen of this compound showed relative specificity for calmodulin kinases.