Immunogens carried in lymphatic fluid drain via afferent vessels into regional lymph nodes and facilitate the efficient induction of appropriate immune responses. The lymphatic system possesses receptors recognizing hyaluronic acid (HA). Covalent conjugates of small-molecule TLR7/8 agonists with HA are entirely devoid of immunostimulatory activity in vitro. In murine models of immunization, however, such conjugates traffic to lymph nodes, where they are "unmasked", releasing the small molecule TLR7/8 agonist from the carrier polysaccharide. The resulting focal immunostimulation is manifested in potent adjuvantic effects with negligible systemic exposure. The efficient delivery of immunogens has been a major challenge in the development of subunit vaccines, and enhancing targeted delivery of immunogens to secondary lymphoid organs might be a promising approach for improving vaccine efficacy, as well as safety.

An important component of vaccine development is the identification of safe and effective adjuvants. We sought to identify transcriptomal signatures of innate immune stimulating molecules using nextgeneration RNA sequencing with the goal of being able to utilize such signatures in identifying novel immunostimulatory compounds with adjuvant activity. The CC family of chemokines, particularly CC chemokines 1, 2, 3, 4, 7, 8, 17, 18, 20, and 23, were broadly upregulated by most Toll-like receptor (TLR) and nucleotide-binding domain and leucine-rich repeat-containing receptors (NLR) stimuli. Extracellular receptors such as TLR2, TLR4 and TLR5 induced the transcription of CXC chemokines including CXCL5, CXCL6 and CXCL8, whereas intracellular receptors such as TLR7 and TLR8 upregulated CXC chemokines 11 and 12. Both TLR1/2 and TLR2/6 agonists induced strong chemokine production in human peripheral blood mononuclear cells. Human skeletal muscle cells and fibroblasts respond with chemokine production only to TLR2/6 agonists, but not TLR1/2 agonists, consistent with strong expression of TLR2 and TLR6, but not of TLR1, in fibroblasts. TLR2/6 stimulated fibroblasts demonstrated functional chemotactic responses to human T cell and natural killer cells subsets. The activation of non-hematopoietic, adventitial cells such as fibroblasts and myocytes may contribute.

Using a multiplexed, reporter gene-based, high-throughput screen, we identified 9-fluoro-7-hydroxy-3-methyl-5-oxo-N-(pyridin-3-ylmethyl)-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinoline-6-carboxamide as a TLR2 agonist. Preliminary structure-activity relationship studies on the carboxamide moiety led to the identification of analogues that induce chemokines and cytokines in a TLR2-dependent manner. These results represent new leads for the development of vaccine adjuvants.

The induction of toll-like receptor 7 (TLR7)-dependent type I interferons (IFN-α/β) from plasmacytoid dendritic cells as well as the production of TLR8-dependent type II interferon (IFN-γ), TNF-α, and IL-12 in myeloid dendritic cells are of importance in generating T helper-1 biased adaptive immune responses. In an effort to identify novel dual TLR7/TLR8-active compounds, we undertook structure-activity relationship studies in pyrimidine 2,4-diamines, focusing on substituents at C5. Several analogues substituted with aminopropyl appendages at C5 displayed dominant TLR8-agonistic activity. N4-Butyl-6-methyl-5-(3-morpholinopropyl)pyrimidine-2,4-diamine was found to be a very potent dual TLR7/TLR8 agonist. Employing novel cytokine reporter cell assays, we verified that potency at TLR7 correlates with IFN-α/β production in human blood, whereas IFN-γ and TNF-α induction is largely TLR8-dependent. Dual TLR7/TLR8 agonists markedly upregulate CD80 expression in multiple dendritic cell subsets, providing insight into the immunological basis for the superior adjuvantic properties of such innate immune stimuli.

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 small-molecule 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.

Activation of human toll-like receptor-8 (TLR8) evokes a distinct cytokine profile favoring the generation of Type 1 helper T cells. A multiplexed high-throughput screen had led to the identification of N(4)-butyl-5-iodo-6-methylpyrimidine-2,4-diamine as a pure TLR8 agonist, and a detailed structure-activity relationship study of this chemotype was undertaken. A butyl substituent at N(4) was optimal, and replacement of the 5-iodo group with chloro, bromo, or fluoro groups led to losses in potency, as did the introduction of aromatic bulk. Drawing from our previous structure-based design, several 5-alkylamino derivatives were evaluated. Significant enhancement of potency was achieved in 5-(4-aminobutyl)-N(4)-butyl-6-methylpyrimidine-2,4-diamine. This compound potently induced Th1-biasing IFN-γ and IL-12 in human blood, but lower levels of the proinflammatory cytokines IL-1β, IL-6, and IL-8. These results suggest that the inflammatory and reactogenic propensities of this compound could be considerably more favorable than other TLR8 agonists under evaluation.

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.

Small-molecule agonists have been identified for TLR7, TLR8, TLR4 and TLR2 thus far, and chemotypes other than those of canonical ligands are yet to be explored for a number of innate immune receptors. The discovery of novel immunostimulatory molecules would enhance the repertoire of tools available for interrogating innate immune effector mechanisms, and provide additional venues for vaccine adjuvant development. A multiplexed, reporter gene-based high-throughput assay capable of detecting agonists of TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1 and NOD2 was utilized in screening 123,943 compounds, in which amphotericin B (AmpB) and nystatin were identified as prominent hits. The polyene antifungal agents act as TLR2- and TLR4-agonists. The TLR4-stimulatory activity of AmpB was similar to that of monophosphoryl lipid A, suggestive of TRIF-biased signaling. The adjuvantic activity of AmpB, at a dose of 100 micrograms, was comparable to several other candidate adjuvants in rabbit models of immunization. These results point to its potential applicability as a safe and effective adjuvant for human vaccines.

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.

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.

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 anticancer 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 tetra(ethylene glycol) (TEG) as a linker. The liposome-based anticancer 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 nonimmunized 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.

Well-defined substrates for the study of oxidative processes are important for the elucidation of the role of DNA damage in the etiology of diseases such as cancer. We have synthesized 3'-modified oligodeoxyribonucleotides (ODNs) using 5'→3' 'reverse' DNA synthesis for the study of 2-deoxyribose oxidative damage to DNA. The modified monomers designed for these studies all share a common feature, they lack the naturally occurring 3'-hydroxyl group found in 2-deoxyribonucleosides. Modified H-phosphonates containing 3'-phenyl selenides as well as saturated and unsaturated sugars were obtained and incorporated in ODNs. These ODNs were used to investigate the fate of C3'-dideoxyribonucleotide radicals in DNA.