TLR4 and TLR7 ligand formulations as vaccine adjuvants

Abstract

Methods for enhancing an immune response in a mammal are provided, as well as compositions comprising a liposome, a TLR4 agonist, and a TLR7 agonist.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Patent Application No. 62 / 818,517, filed March 14, 2019, the disclosure of which is incorporated herein by reference.

[0002] [Government Rights Statement] This invention was made with Government support under Grant No. HHSN272200900034C awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] 〔background〕 The most effective way to protect individuals against the insidious threats of many infectious diseases is through vaccination. Effective vaccination requires the use of an antigen that induces an immune response in the host that can provide subsequent protection against the particular infectious disease agent for which the vaccine is specific. Thus, the vaccine antigen must be sufficiently immunogenic to induce a level of immune response - humoral and / or cell-mediated - in the host that is protective. An infectious pathogen of global concern is the influenza virus. Seasonal influenza viruses cause annual epidemics and are responsible for 250-500,000 deaths worldwide (WHO). Last winter, more than 80,000 people died in the United States alone. In addition, new pandemics occur from time to time, causing millions of deaths and posing a serious global threat. Particularly vulnerable to these threats are high-risk populations such as the elderly, newborns, and immunocompromised individuals. Vaccination against seasonal influenza can be moderately effective if matched to the circulating viral strains of that season. However, because influenza viruses are constantly changing (antigenic drift), it is difficult to predict which subtypes and strains of virus will circulate in the next influenza season or the next pandemic and to ensure sufficient time (approximately six months) for the manufacture and distribution of conventional vaccines.

[0004] These conventional vaccines are typically based on influenza hemagglutinin (HA) proteins, specifically antigens associated with the globular head domain of the protein. This highly immunogenic head domain is diverse across influenza virus strains and subtypes. Thus, an immune response to one globular head domain subtype is limited to that particular head domain and may not provide an adequate immune response to a virus strain with a different head domain. Influenza HA antigens derived from stem or stalk domains of proteins that are more highly conserved across virus strains are generally much less immunogenic than the head domain antigens that typically predominate in conventional vaccines. Thus, there is a need to improve the immunogenicity of these HA stalk antigens to a level that will generate a sufficient immune response in the host, resulting in an immune response against multiple influenza strains.

[0005] Summary of the Invention The successful use of appropriate adjuvant combination formulations in vaccines against infectious pathogens of global importance, such as influenza virus, would potentially represent a major advance in medicine, broadening and enhancing individuals' defenses against the ever-changing threat of these viral pathogens.

[0006] The present disclosure provides a process for formulating a combination of TLR4 agonist and TLR7 agonist, and adjuvant in the same liposomal nanoparticle provides several advantages over a mixed combination of separate formulated and unformulated agonists. The formulated combination can have a specific ratio of TLR4 to TLR7 in the nanoparticle for desired immune activity. Based on the data generated by the various combination ratios of the compounds, each compound was formulated alone and in combination. The formulated and unformulated combinations were compared side-by-side, mixed and combined in the same particle. The results of the immunization study showed that the compounds combined in a specific ratio in liposomes provided greater and broader immune activity than either compound alone, and the formulated combination was better than the unformulated combination. The antigens used were OVA and inactivated influenza virus.

[0007] As disclosed herein, 2B182C (an exemplary TLR4 agonist) and 1V270 (an exemplary TLR7 agonist) were formulated together in one formulation, and immunization studies were conducted in mice. Based on the data generated by various combination ratios of compounds, each compound was formulated alone and in combination. Side-by-side comparisons were made between formulated and unformulated combinations, as well as mixing and combinations in the same particle. The results of the immunization studies showed that the compounds combined at a specific ratio in liposomes provided greater and broader immune activity than either compound alone, and the formulated combination was better than the unformulated combination. The antigens used were OVA and inactivated influenza virus.

[0008] In one embodiment, the present disclosure provides a method for enhancing immune response in a mammal, comprising administering an effective amount of a TLR4 agonist and a TLR7 agonist to a mammal in need thereof. In one embodiment, the TLR4 agonist and the TLR7 agonist are co-administered. In one embodiment, the TLR4 agonist and the TLR7 agonist are administered in a liposomal formulation. In one embodiment, the TLR4 agonist and the TLR7 agonist are present in separate liposomal formulations. In one embodiment, the TLR4 agonist has formula (II). In one embodiment, the TLR7 agonist has formula (I). In one embodiment, one or more immunogens (antigens) are administered, for example, simultaneously with an adjuvant, and optionally in the same formulation as the adjuvant. In one embodiment, the immunogen is a microbial immunogen. In one embodiment, the microorganism is a virus, such as influenza or chickenpox, or a bacterium. In one embodiment, the mammal is a human. In one embodiment, the amount of the TLR7 agonist is about 0.01 to 100 nmol, about 0.1 to 10 nmol, or about 100 nmol to about 1000 nmol. In one embodiment, the amount of the TLR4 agonist is about 2 to 20 umol, about 20 nmol to 2 umol, or about 2 umol to about 100 umol. In one embodiment, the ratio of the TLR7 agonist to the TLR4 agonist is about 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200. In one embodiment, the formulation is injected. In one embodiment, the liposome formulation comprises DOPC, cholesterol, or a combination thereof.

[0009] Also provided is a pharmaceutical formulation comprising a liposome, a TLR4 agonist, and a TLR7 agonist, for example, the liposome comprises DOPC, cholesterol, or a combination thereof. In one embodiment, the amount of the TLR7 agonist is about 0.01-100 nmol, about 0.1-10 nmol, or about 100 nmol to about 1000 nmol, and the amount of the TLR4 agonist is about 2 nmol to 20 umol, about 20 nmol to 2 umol, or about 2 umol to about 100 umol, or the ratio of the TLR7 agonist to the TLR4 agonist is about 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200.

[0010] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Exemplary liposome formulations.

[0011] Figure 2: In vitro immunostimulatory activity of 1V270 (1 μM), 2B182C (200 μM), or a combination of 1V270 (1 μM) and 2B182C (200 mM) in DMSO or liposomal formulation. Murine bone marrow-derived dendritic cells from wild-type C57BL / 6 mice were incubated with 1V270 (1 μM), 2B182C (200 μM), or a combination of 1V270 (1 μM) and 2B182C (200 mM) in DMSO or liposomal formulation for 18 hours. IL-6 release in culture supernatants was measured by ELISA.

[0012] Figure 3: Liposomal formulation reduces TLR4-independent cytotoxicity. Mouse bone marrow-derived dendritic cells from wild-type C57BL / 6 mice or TLR4-deficient mice (C57BL / 6 background) were incubated with 1V270 (1 μM), 2B182C (200 μM), or a combination of 1V270 (1 μM) and 2B182C (200 mM) in DMSO or liposomal formulation for 18 h. Cell viability was assessed by MTT assay.

[0013] FIG. 4: Exemplary experimental protocol.

[0014] [Figure 5] Anti-HA IgG1 and IgG2a levels in response to formulations.

[0015] [Figure 6] IgG2a / IgG1 ratio for each formulation.

[0016] [Figure 7] Gating strategy for GC cells and plasmablasts.

[0017] Figure 8: Cell types induced by administration of 1V270 and / or 2B182c or AddaVax. The combination of formulated 1V270 and 2B182c significantly increased the number of GC B cells and plasmablasts.

[0018] FIG. 9: Anti-HA IgG levels measured by ELISA or as BCR-seq induced by administration of 1V270 and / or 2B182c or AddaVax. IgG2a production was strongly increased by the combination treatment, and Th1 responses were induced by formulated 2B182c.

[0019] FIG. 10. BCR diversity increased with combination treatment.

[0020] Figure 11: TCR clonality following administration of antigen with 1V270 and / or 2B182c or Addavax. TCR clonality increased following 2B182c treatment and Addavax.

[0021] FIG. 12: BCR diversity and TCR clonality following administration of 1V270 and / or 2B182c or AddaVax. BCR diversity was increased by combination treatment, and TCR clonality was increased following 2B182c treatment and Addavax.

[0022] [Figure 13] Clonal similarity.

[0023] [Figure 14] Shared clones.

[0024] [Figure 15] Cluster analysis.

[0025] [Figure 16] Number of clusters with similar sequences to known antibodies against influenza.

[0026] Figure 17: Analysis of cytotoxicity and IL-12 secretion. Liposomal adjuvants induced IL-12 secretion in BMDCs with lower cytotoxicity.

[0027] FIG. 18. Anti-NA IgG1 and IgG2a analysis following administration of 1V270 and / or 2B182c (unformulated and formulated) or AddaVax.

[0028] [Figures 19A-19B] Liposomal formulations of 2B182c and 1V270 bias the immune response towards a Th1 response. (A) BALB / c mice were immunized with inactivated Cal 2009 H1N1 influenza virus (10 μg / injection) mixed with TLR4 and / or TLR7 ligands in DMSO (D) or liposomal (L) formulations on days 0 and 28. Serum was collected on day 28. HA- or NA-specific IgG1 and IgG2a were determined by ELISA. (B) Th1 / TH2 balance was assessed by the IgG2a / IgG1 ratio using the Mann-Whitney test. * P < 0.05, ** P<0.001.

[0029] [Figures 20A-20C] Combination adjuvant treatment with liposomal 2B182c and liposomal 1V270 increased the numbers of germinal center B cells and plasmablasts in draining lymph nodes. (A) Experimental protocol. (B) Gating strategy for flow cytometry data. (C) B cells, germinal center B cells (CD3 - CD19 + CD95 + GL7 + ), and plasmablasts (CD3 - CD19 + CD138 +) was calculated. BL; blank liposomes. The Kruskal-Wallis test with Dunn's post-hoc test showed that, compared with antigen + BL, * p<0.05, ** p<0.01, *** ;p<0.001.

[0030] [Figures 21A-21B] 2B182C is effective against both human (A) and mouse (B) TLR4 at lower concentrations. HEK TLR reporter cells (HEK-Blue TM hTLR4 and HEK-Blue TM mTLR4) were treated with compounds 1Z105 and 2B182C (2-fold serial dilutions from 10 μM) for 20 h. NF-kB-inducible NF-kB SEAP levels in culture supernatants were assessed according to the manufacturer's protocol.

[0031] [Figures 22A-22C] 200 nmol / injection of 2B182c induced higher levels of antigen-specific IgG1 and anti-NA IgG2a. (A) Experimental protocol for comparing two TLR agonists 1Z105 and 2B182C. BALB / c mice (n=5 / group) were immunized with IIAV (10 μg / injection) plus TLR4 agonists 1Z105 or 2B182C (40 and 200 nmol / injection) intramuscularly in both hind legs on days 0 and 21. Blood was collected on day 28 and sera were evaluated for antibodies against hemagglutinin (HA) and neuraminidase (NA) by ELISA. 10% DMSO was used as vehicle. (B) Anti-HA IgG1 and anti-NA IgG1 antibodies. (C) Anti-HA IgG2a and anti-NA IgG2a antibodies. In each box plot, the line within the box represents the median, the boundaries are the upper and lower quartiles, and the bars indicate the minimum and maximum values. * P < 0.05, ** P<0.001.

[0032] [Figures 23A-23C] Combination of 2B182C and the TLR7 agonist 1V270 increased both antigen-specific IgG1 and IgG2a. (A-C) BALB / C mice (n=5-6) were immunized with IIAV and adjuvant as shown in Figure 2A. AddaVax, a formulation similar to MF59, was used. TM was used as a positive control. Anti-HA IgG1 and anti-NA IgG1 production (A), anti-HA IgG2a and anti-NA IgG2a production (B) were determined by ELISA. In each box plot, the line within the box represents the median, the boundaries are the upper and lower quartiles, and the bars indicate the minimum and maximum values. By Kruskal-Wallis test with Dunn's post-hoc test, * P < 0.05, ** P < 0.01, *** P<0.001. Comparisons were made between no antigen and all four groups except AddaVax (all pairs). (C) Anti-HA IgG1 and IgG2a levels (normalized to vehicle) induced by all combination treatments are shown by average of 5-11 mice / group. Each dot represents an individual animal. The solid black line indicates IgG2a / IgG1=1. All animals immunized with the combination of 1V270 and 2B182C were distributed above IgG2a / IgG1=1. This suggests that the immune balance in these mice was biased towards a Th1 immune response.

[0033] [Figures 24A-24B] Antigen-specific IgM production on day 28. (A and B) BALB / c mice (n=5-6) were immunized with IIAV (10 μg / injection) and adjuvants as shown in Figure 2A. Antigen-specific IgM levels were measured by ELISA. (A) Anti-HA IgM and anti-NA IgM production induced by TLR4 agonists 1Z105 or 2B182C (40 and 200 nmol / injection). (B) The combination of TLR7 agonist 1V270 (1 nmol / injection) with TLR4 agonists 1Z105 or 2B182C (200 nmol / injection) had little effect on antigen-specific IgM induction. Kruskal-Wallis test with Dunn's post-hoc test showed that the levels of IgM were significantly higher than those of the control group. * P<0.05.

[0034] [Figures 25A-25B] Liposomal 1V270 and liposomal 2B182C induced comparable levels of IL-12 release with lower toxicity. (A) IL-12 secretion levels. (B) % viability. Mouse primary BMDCs were treated with 1V270 (0.0625uM) and 2B182C (12.5uM). The 1V270 / 2B182c ratio was kept at 1:200, which was judged as the best ratio in Figure 3. After overnight incubation, IL-12 levels in the culture supernatants were examined by ELISA, and cell viability was evaluated by MTT assay. One-tailed unpaired t-test with Welch correction showed that the DMSO formulation (D) vs. the liposomal formulation (L) of each compound was significantly different from that of the control. * P < 0.05, ** P<0.01.

[0035] FIG. 25C: Histological analysis of local immune cell infiltration after injection of combined adjuvants. BALB / c mice were intramuscularly injected with liposomal formulations of 1V270 (1 nmol / injection), 2B182C (200 nmol / injection), or a combination of 1V270 (1 nmol / injection) and 2B182C (200 nmol / injection). Tissues were harvested, fixed, and embedded in paraffin blocks. 10 μm sections were stained with H&E. Low and high magnification images were obtained using 20× and 40× objectives, respectively. Scale bars in low and high magnification images indicate 50 μm and 20 μm, respectively.

[0036] FIG. 25D: Vehicle, 1V270, 2B182C, 1V270+2B182C were injected intramuscularly into BALB / c mice (n=5 / group) with DMSO or liposomal formulations (1 nmol / injection of 1V270 and 200 nmol / injection of 2B182C in a volume of 50 μL). AddaVax TM (25 μL / injection) was used as a positive control. Serum was collected after 2 and 24 hours and examined for IL-12p40, TNF, and KC secretion by Luminex multiplex cytokine assay (A). Data are presented as mean ± SEM. Two-tailed Mann-Whitney U test showed that * P < 0.05, ** P<0.01. Kruskal-Wallis test with Dunn's post-hoc test was used to compare the four groups (vehicle, 1V270, 2B182C, 1V270+2B182C in the same formulation). + P < 0.05, ++ P<0.01.

[0037] [Figures 26A-26D] Liposomal 1V270 and liposomal 2B182C synergistically enhanced anti-HA IgG1 and IgG2a production, and anti-NA IgG1 and IgG2a production. (AC) As shown in Figure 22A, BALB / c mice (n=5 / group) were immunized by intramuscular injection with IIAV (10 μg / injection) and formulated adjuvants on days 0 and 21. Liposomal TLR7 agonist 1V270 (Lipo-1V270, 1 nmol / injection), liposomal TLR4 agonist 2B182C (Lipo-2B182C, 200 nmol / injection), and liposomal complex adjuvant of 1V270 and 2B182C (Lipo-1V270+2B182C, 1 nmol / injection+200 nmol / injection) were injected. The vehicle is 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol (DOPC / Chol, control liposomes). AddaVax TM was used as a positive control. Serum was collected on day 28 and HA- or NA-specific IgG1, IgG2a, and total IgG were determined by ELISA. * P < 0.05 and ** P<0.01. All four groups were compared except for No Antigen and AddaVax (all pairs). Data are representative of two independent experiments with similar results.

[0038] Figure 27: Antigen-specific IgM levels induced by formulated adjuvants. BALB / c mice (n=5 / group) were immunized intramuscularly with IIAV (10 μg / injection) with formulated adjuvants as shown in Figure 2A on days 0 and 21. The liposomal TLR7 agonist 1V270 (Lipo-1V270, 1 nmol / injection), the liposomal TLR4 agonist 2B182C (Lipo-2B182C, 200 nmol / injection), and the composite liposomal adjuvant of 1V270 and 2B182C (Lipo-1V270+2B182C, 1 nmol / injection+200 nmol / injection) were injected. The vehicle was 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol (DOPC / Chol, control liposome). AddaVax TM was used as a positive control. Serum was collected on day 28 and tested for IgM specific for HA or NA. * P<0.05. Four treatments were compared except for No antigen and AddaVax (all pairs). Data are representative of two independent experiments with similar results.

[0039] [Figures 28A-28C] Formulated conjugate adjuvants increased Tfh and antibody secreting cells. (A) BALB / c mice (n=4-5 / group) were vaccinated with IIAV (10 μg / injection) with 1V270 (1 nmol / injection) and / or 2B182C (200 nmol / injection) in a total volume of 50 μL on days 0 and 21. On day 28, lymphocytes in inguinal lymph nodes were harvested for FACS analysis. Gating strategies for Tfh cells (CD3+ CD4+ PD-1+ CXCR5+), GC B cells (CD3- CD19+ CD95+ GL7+), plasmablasts (CD3- CD19+ CD138+), and plasma cells (CD3- CD19- CD138+) are shown. (B) % Tfh cells, GC B cells, plasmablasts, and plasma cells in live cells. Bars indicate mean ± SEM. Kruskal-Wallis test with Dunn's post-hoc test * P < 0.05 and** P<0.01. All four conditions except AddaVax were compared (all pairs).

[0040] [Figure 29A-29B] Formulated composite adjuvants increased Tfh and antibody secreting cells. BALB / c mice (n=4-5 / group) were vaccinated with IIAV (10 μg / injection) with 1V270 (1 nmol / injection) and / or 2B182C (200 nmol / injection) in a total volume of 50 μL on days 0 and 21. On day 28, lymphocytes in the inguinal lymph nodes were harvested for FACS analysis (Figure 5A). The gating strategy for Tfh cells (CD3+ CD4+ PD-1+ CXCR5+), GC B cells (CD3- CD19+ CD95+ GL7+), plasmablasts (CD3- CD19+ CD138+), and plasma cells (CD3- CD19- CD138+) is shown in Figure 5B. (A) Numbers of Tfh cells, GC B cells, plasmablasts, and plasma cells. (B) Total number of cells. Bars indicate mean ± SEM. Kruskal-Wallis test with Dunn's post-hoc test showed that * P < 0.05 and ** P<0.01 (all pairs).

[0041] Figures 30A-30C: Formulated combination of 1V270 and 2B182C. (A and B) BALB / c mice were vaccinated with IIAV with formulated adjuvant on days 0 and 21, and inguinal lymph nodes were harvested on day 28 for BCR repertoire analysis. (A) BCR diversity for total IGH, IGHG1, and IGHG2A. (B) Similarity analysis. Jaccard index is shown. (C) TCR clonality as indicated by "1-pielou index" for TCRα and TCRβ. Bars indicate mean ± SEM. Kruskal-Wallis test with Dunn's post-hoc test showed that the mean (compared to liposomes) was significantly higher than that of liposomes. * P < 0.05 and ** P<0.01.

[0042] [Figures 31A-31I] Lipo-2B182C and Lipo-1V270+2B182C protect mice against homologous influenza virus. (A) Experimental schedule of homologous influenza virus challenge. (B) Mean body weight change is shown by % initial body weight. One-way ANOVA with Dunnett's post-hoc test showed that * P < 0.05, ** P<0.01. (C) Survival of mice after challenge with the homologous virus (H1N1pdm). Kaplan-Meier curves with log-rank test are shown. Lung virus titers (D) and cytokine levels in lung fluid (E) were assessed. Lung lavage was performed on days 3 and 6. The Kruskal-Wallis test with Dunn's post-hoc test showed that the survival rate of mice (compared to liposomes) was 1.2%. ** P < 0.01. (F) Relationship between lung virus titers and the proinflammatory cytokines MCP-1 (left) and IL-6 (right). Spearman rank correlation test (MCP-1; ** P<0.0001, Spearman r=0.83, IL‐6; *** P<0.0001, Spearman r=0.79). HI titers (G) and VN titers against the same virus (H). Kruskal-Wallis test with Dunn's post hoc test * P < 0.01 and *** P<0.001 (all pairs). (I) Relationship between VN titer and lung virus titer. Each dot represents the VN titer and lung virus titer in the same animal. ** P<0.01, Spearman rank correlation test, Spearman r=-0.59.

[0043] Figures 32A-32C: Heterologous challenge with H3N2 virus. BALB / c mice were immunized with IIAV(H1N1) plus formulated adjuvant as described in Figure 31A and challenged intranasally with the heterologous virus A / Victoria3 / 75(H3N2). (A) Weight loss was monitored. No significance was detected by one-way ANOVA. (B) Survival rate of mice after heterologous virus challenge. Kaplan-Meier curves with log-rank test (ns) are shown. (C) Lung virus titers at days 3 and 6. No significance was detected by Kruskal-Wallis test.

[0044] [Figures 33A-33G] A and E) Protocol. B-C and F-G) Body weight and survival over time following infection with A / PuertoRico / 8 / 1934 or B / Florida / 04 / in mice treated with 1V270 and / or 2B182C or AddaVax. D) IgG2a / IgG1 ratio in mice treated with 1V270 and / or 2B182c or AddaVax.

[0045] [Figures 34A-34B] A) Anti-HA IgG1, anti-HA IgG2a, and anti-HA IgM in mice treated with 1V270, 1Z105, 2B182c, or AddaVax. B) Anti-NA IgG1, anti-NA IgG2a, and anti-NA IgM in mice treated with 1V270, 1Z105, 2B182c, or AddaVax.

[0046] [Figures 35A-35F] A and B) Anti-HA and anti-NA IgG1, CD) Anti-HA and anti-NA IgG2a, and EF) Anti-HA and anti-NA IgM in mice treated with 1V270, 1Z105, 2B182c, or AddaVax. B) Anti-NA IgG1, anti-NA IgG2a, and anti-NA IgM in mice treated with 1V270, 1Z105, 2B182c, or AddaVax.

[0047] [Figures 36A-36B] Anti-HA IgG2a and IgG1 in mice administered different doses of 1V270, 1Z105, 2B182c, or their combinations.

[0048] FIG. 37. Schematic diagrams of various liposomes and exemplary protocols.

[0049] [Figure 38A-38B] ELISA using peptide array of HA of A / California / 04 / 2009(H1N1)pdm. BALB / c mice (n=5-10) were immunized with IIAV plus Lipo-Veh (blank liposomes), Lipo-1V270, Lipo-2B182C, Lipo-(1V270+2B182C) (coencapsulated combination), or (Lipo-1V270)+(Lipo-2B182C) (mixed combination) on days 0 and 21, and bled on day 28. Peptide array of HA of A / California / 04 / 2009(H1N1)pdm (NR-15433) was obtained from BEI Resources. Five groups of peptides were pooled to generate 28 peptide pools. (A) OD of ELISA results 405-570nm (B) Heatmap of peptide pools and mice. Each row and column represents a peptide pool and mouse, respectively. (B) Statistical analysis was performed on the average of 28 peptide pools in individual mice. ** P < 0.01, *** P<0.0001, Kruskal-Wallis test with Dunn's post-hoc test. +P<0.05, Mann-Whitney test.

[0050] [Figure 39A-39D] ELISA for antibody cross-reactivity. BALB / c mice (n=5 / group) were immunized with IIAV plus Lipo-Veh, Lipo-1V270, Lipo-2B182C, Lipo-(1V270+2B182C), or (Lipo-1V270)+(Lipo-2B182C) on days 0 and 21 and bled on day 28. Sera were serially diluted (1:100 to 1:409600) and assessed by ELISA for total IgG levels against Puerto RicoH1N1, H11N9, H12N5, H7N7, and H3N2 HAs, and H5N1, H10N8, H3N2, and H7N7 NAs. (A) Phylogenetic relationship of the HAs of influenza A viruses used in this study. The amino acid sequences of proteins used in ELISA were aligned by the MUSCLE algorithm using the Influenza Research Database (https: / / www.fludb.org / brc / home.spg?decorator=influenza). Phylogenetic trees were constructed by the neighbor-joining method using MEGAX software (https: / / www.megasoftware.net / ). (B) Total IgG titer curves against HA of H1N1, H11N9, H12N5, H3N2, and H7N7. (C) Phylogenetic relationships of NA. (D) Total IgG titer curves against NA of H5N1, H10N8, H3N2, and H7N7. Sera were diluted 1:4 starting from 100-fold to 409600-fold, and total IgG levels were assessed by ELISA. Data shown are mean ± SEM.

[0051] [Figures 40A-40B] Lipo-(1V270+2B182C) induced cross-reactive antibodies. (A and B) BALB / c (n=5 / group) mice were immunized with IIAV [A / California / 2009(H1N1)pdm09] plus Lipo-Veh, Lipo-1V270, Lipo-2B182C, Lipo-(1V270+2B182C), or (Lipo-1V270)+(Lipo-2B182C) on days 0 and 21 and bled on day 28. Sera were serially diluted (1:100 to 1:409600) and total IgG levels were assessed by ELISA against the HA of Puerto RicoH1N1, H11N9, H12N5, H7N7, and H3N2, and the NA of H5N1, H10N8, H3N2, and H7N7. The geometric mean of the total IgG titer curves of individual mice calculated using prism 5 is shown. The total IgG titer curves and phylogenetic relationships of the HA proteins used in this study are shown above. * P < 0.05, ** P<0.01, Kruskal-Wallis test with Dunn's post-hoc test. +P<0.05, ++P<0.01, Mann-Whitney test.

[0052] FIG. 41. Exemplary TLR4 and TLR7 agonists.

[0053] Detailed Description The use of adjuvants in vaccines is a well-established method to promote stronger immune responses to weakly immunogenic antigens. In addition, adjuvants can also enhance and potentially broaden immune responses by promoting the immunogenicity of weakly immunogenic antigens. Currently, only a few adjuvants are approved for use in vaccines (O'Hagan, et al. doi: 10.1016 / j.vaccine.2015.01.088). Furthermore, the majority of existing vaccines contain a single adjuvant, and recent evidence suggests that this is not sufficient to induce protective immunity against many emerging infectious diseases (Underhill, doi: 10.1111 / j.1600-065X.2007.00548.x).

[0054] The use of combinations of TLR agonists as adjuvants has often resulted in an overall boost in immune responses. However, in the case of infectious disease vaccines such as influenza, the boosting of Th1 (cell-mediated) or the biasing of responses towards Th1 types comes at the expense of Th2 (humoral or antibody) types. In fact, despite an increased Th1 response, the production of protective Th2 antibodies can be insufficient, and for influenza infection, a certain level of protective antibody titer is thought to be the primary factor for providing effective protection via immunization.

[0055] In this disclosure, it was found that the ratio of combination of TLR4 / TLR7 agonists in a single nanoparticle formulation not only enhances the overall immune response to antigens, but also provides sufficient protective antibody generation for effective protection against lethal viral challenge in mice. The immunological status of humans will be quite different from that of mice. Mice are not commonly exposed to influenza-like antigens, whereas humans have been exposed to influenza antigens for many years, usually through both natural and vaccine exposure. The same is true for other infectious pathogens, such as chickenpox (varicella zoster), which can later manifest as shingles in humans.

[0056] Immunization with one antigen is known to prevent a robust immune response against a second similar antigen, which may be due to 1) epitope exclusion (pre-existing antibodies, especially mucosal IgA, shield the vaccine from all antigen-presenting cells); 2) reduced dendritic cell (DC) access (memory B cells internalize the new vaccine, reducing DC access and activation, as well as T cell immunization); and 3) T cell competition (memory B cells are activated, consuming cytokines, cofactors, and capturing T cells that can react with antigen-loaded DCs).

[0057] The present disclosure overcomes these burdens by 1) encapsulating the vaccine in liposomal nanoparticles that preferentially deliver the vaccine to DCs, and 2) activating DCs using combined TLR agonists in a specific ratio that increases the diversity of the number of activated T cells against vaccine antigens.The present invention discloses the inventors' discovery that formulating a combination of TLR4 agonist and TLR7 agonist in the same liposomal nanoparticle as an adjuvant offers several advantages over a mixed combination of separately formulated and unformulated agonists.The formulated combination can have a specific ratio of TLR4 to TLR7 in the nanoparticle for immune activity.

[0058] The advantages of these combinations at this ratio include: 1) enhanced activation for DMSO formulations providing better Th1 and Th2 immune responses; 2) lower toxicity for DMSO formulations; 3) shielding of antigens (for vaccine use) from B cells and IgA in hyperimmune individuals, particularly for mucosal influenza immunization, and allowing dendritic cells to present important epitopes for an effective protective response; and / or 4) broadening the immune response to include responses to less immunogenic antigens, such as the case of the HA stalk antigen in influenza, resulting in a more universal vaccine.

[0059] The use of combinations of TLR agonists as adjuvants has often resulted in an overall enhancement of the immune response. However, in the case of vaccines for infectious diseases such as influenza, the enhancement of Th1 (cell-mediated) or the bias of the response towards Th1 type comes at the expense of Th2 (humoral or antibody) type. Indeed, despite an increased Th1 response, the production of protective Th2 antibodies can be insufficient, and for influenza infections, a certain level of protective antibody titer is thought to be the primary factor for providing effective protection via immunization.

[0060] In the present invention, the ratio of the combination of TLR4 / TLR7 agonists in a single nanoparticle formulation was found to not only enhance the overall immune response to the antigen, but also provide sufficient protective antibody generation for effective protection against lethal viral challenge in mice. The immunological status of humans will be quite different from that of mice. Mice are not generally exposed to influenza-like antigens, whereas humans have been exposed to influenza antigens for many years, usually through both natural and vaccine exposure. The same is true for other infectious pathogens, such as chickenpox (varicella zoster), which can later manifest as shingles in humans.

[0061] Immunization with one antigen is known to prevent a robust immune response against a second, similar antigen, which may be due to 1) epitope exclusion (pre-existing antibodies, especially mucosal IgA, shield the vaccine from all antigen-presenting cells); 2) reduced dendritic cell (DC) access (memory B cells internalize the new vaccine, reducing DC access and activation, as well as T cell immunization); and 3) T cell competition (memory B cells are activated, consuming cytokines, cofactors, and capturing T cells that can react with antigen-loaded DCs).

[0062] The present disclosure overcomes these burdens by 1) encapsulating the vaccine in liposomal nanoparticles that deliver the vaccine preferentially to DCs, and 2) activating DCs with combinatorial TLR agonists in specific ratios that increase the diversity of numbers of activated T cells against the vaccine antigen.

[0063] [Definition] A composition is composed of "substantially all" of a particular compound, or a particular form of a compound (e.g., an isomer) when the composition comprises at least about 90% of a particular composition by weight, as well as at least about 95%, 99%, and 99.9%. A composition comprises a "mixture" of compounds or forms of the same compound when each compound (e.g., an isomer) represents at least about 10% of the composition by weight. The TLR7 agonist or conjugate of the present invention can be prepared as an acid salt or base salt, as well as in free acid or free base form. In solution, certain of the compounds of the present invention can exist as zwitterions, where the counterion is provided by the solvent molecule itself or from other ions dissolved or suspended in the solvent.

[0064] The term "Toll-like receptor agonist" (TLR agonist) refers to a molecule that binds to a TLR. Synthetic TLR agonists are chemical compounds designed to bind to a TLR and activate the receptor.

[0065] It is to be understood that within the scope of the present invention, compounds of formula (I) or formula (II), or salts thereof, may exhibit the phenomenon of tautomerism, whereby two compounds form a covalent bond that can be easily interconverted by exchanging hydrogen atoms between the two atoms. Tautomeric compounds exist in a transfer equilibrium with each other, so they can be considered as different isomeric forms of the same compound. It is to be understood that the formula drawings within this specification may represent only one of the possible tautomeric forms. However, it is also to be understood that the present invention encompasses any tautomeric form and is not merely limited to any one tautomeric form utilized within the formula drawings. It is to be understood that the formula drawings within this specification may represent only one of the possible tautomeric forms, and that the specification encompasses all possible tautomeric forms of the depicted compounds, not just those that have been conveniently illustrated in the specification. For example, tautomerism may be represented by pyrazolyl groups linked as indicated by wavy lines. It is clear that both of the substituents are referred to as 4-pyrazolyl groups, but different nitrogen atoms have hydrogen atoms in each structure.

[0066] [ka]

[0067] Such tautomerism can also occur in substituted pyrazoles, such as 3-methyl, 5-methyl, or 3,5-dimethylpyrazole. Another example of tautomerism is amide-imide (or, if cyclic, lactam-lactim) tautomerism, as found in heterocyclic compounds that have a ring oxygen atom adjacent to a ring nitrogen atom. For example, the equilibrium:

[0068] [ka]

[0069] is an example of tautomerism. Thus, a structure shown herein as one tautomer is intended to include the other tautomers as well.

[0070] [Optical isomerism] It will be understood that when the compounds of the invention contain one or more chiral centers, the compounds may exist and be isolated in pure enantiomeric or diastereomeric forms, or as racemic mixtures.Accordingly, the present invention includes any possible enantiomers, diastereomers, racemates, or mixtures thereof of the compounds of the invention.

[0071] Isomers resulting from the presence of chiral centers include a pair of non-superimposable isomers called "enantiomers". Single enantiomers of a pure compound are optically active, i.e., they are capable of rotating the plane of plane polarized light. Single enantiomers are designated according to the Cahn-Ingold-Prelog system. The priority of the substituents is ranked based on atomic weight, with higher atomic weights having higher priority, as determined by a systematic procedure. Once the priority of the four groups has been determined, the molecule is oriented so that the lowest ranking group is pointed away from the observer. The molecule is then designated (R) if the descending order of the other groups proceeds clockwise, and (S) if the descending order of the other groups proceeds counterclockwise. In Scheme 14, for example, the Cahn-Ingold-Prelog ranking rule is A>B>C>D, with the lowest ranking atom D pointing away from the observer.

[0072] [ka]

[0073] The present invention is meant to encompass diastereomers as well as their racemic and resolved, diastereomerically and enantiomerically pure forms and their salts. Diastereomeric pairs can be separated by known separation techniques, including normal and reverse phase chromatography, and crystallization.

[0074] "Isolated optical isomer" means a compound that is substantially purified from the corresponding optical isomer of the same formula. In one embodiment, the isolated isomer is at least about 80% by weight, e.g., at least 90%, 98%, or 99% pure by weight.

[0075] The isolated optical isomers can be purified from the racemic mixture by well-known chiral separation techniques. According to one such method, the racemic mixture of the compounds of the present invention or its chiral intermediates are separated into optical isomers of 99% purity by weight by HPLC using a suitable chiral column, such as one of the series of DAICEL® CHIRALPAK® family columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). The column is operated according to the manufacturer's instructions.

[0076] The phrase "pharmacologically acceptable" is used herein to refer to compounds, ingredients, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0077] As used herein, "pharmaceutically acceptable salt" refers to the derivative of the disclosed compound, which the parent compound is modified by making its acid salt or base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues (e.g., amines); alkali or organic salts of acidic residues (e.g., carboxylic acids). Pharmaceutically acceptable salts include the conventional non-toxic salts or quaternary ammonium salts of the parent compound, for example, formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like); and the salts prepared from organic acids (e.g., acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, behenic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like).

[0078] The pharma- ceutically acceptable salts of the compounds useful in the present invention can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid form or free base form of these compounds with stoichiometric amounts of appropriate base or acid in water or in an organic solvent or in the mixture of the two.Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile can be used.A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985), the disclosure of which is incorporated herein by reference.

[0079] The compounds of the formulas described herein may be solvates, and in some embodiments may be hydrates. The term "solvate" refers to a solid compound with one or more solvent molecules bound to the solid structure. When a compound is crystallized from a solvent, a solvate may be formed. When solidification causes one or more solvent molecules to become an integral part of the solid crystal matrix, a solvate is formed. The compounds of the formulas described herein may be solvates, for example, ethanol solvates. Another type of solvate is a hydrate. "Hydrate" also refers to a solid compound with one or more water molecules intimately bound to the solid or crystal structure at the molecular level. When a compound is solidified or crystallized in water, a hydrate may be formed, where one or more water molecules become an integral part of the solid crystal matrix.

[0080] The following definitions are used unless otherwise stated; halo or halogen is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc. refer to both straight chain and branched groups, although references to individual radicals such as "propyl" embrace only straight chain radicals; branched chain isomers such as "isopropyl" are specifically referred to. Aryl refers to a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Het may be heteroaryl (including radicals bonded through ring carbons of a monocyclic aromatic ring containing 5 or 6 ring atoms of carbon and 1 to 4 heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X), where X is absent or is an ortho-fused bicyclic heterocyclic radical of about 8 to 10 ring atoms derived therefrom, particularly benz derivatives, or derived by fusing thereto a propylene, trimethylene, or tetramethylene diradical).

[0081] It will be understood by those skilled in the art that the compounds of the present invention having chiral centers can exist and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It will be understood that the present invention encompasses any racemic, optically active, polymorphic, or stereoisomeric form, or mixtures thereof, of the compounds of the present invention having the beneficial properties described herein, and that methods for preparing optically active forms (e.g., by resolution of racemic forms by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using chiral stationary phases) and for determining agonist activity using standard tests described herein or other similar tests known in the art are well known in the art. It will also be understood by those skilled in the art that the compounds described herein include their various tautomers, which can exist in various equilibrium with each other.

[0082] As used herein, the terms "treat" and "treating" refer to (i) preventing a pathological condition from occurring (e.g., prophylaxis); (ii) inhibiting or arresting the progression of a pathological condition; (iii) relieving a pathological condition; and / or (iv) ameliorating, alleviating, lessening, and removing symptoms of a condition. The candidate molecules or compounds described herein may be present in a formulation or medicament in an amount that can produce a biological effect or that can result in, for example, ameliorating, alleviating, lessening, relieving, diminishing, or removing symptoms of a disease. The terms may also refer to reducing or stopping the rate of cell proliferation (e.g., slowing or halting tumor growth) or reducing the number of proliferating cancer cells (e.g., removing part or all of a tumor). These terms are also applicable to reducing the titer of a microorganism (microbe) in a system (e.g., a cell, tissue, or subject) infected with a microbe, reducing the rate of microbial reproduction, reducing the number or effect of symptoms associated with a microbial infection, and / or removing detectable amounts of a microbe from the system. Examples of microbes include, but are not limited to, viruses, bacteria, and fungi.

[0083] The term "therapeutically effective amount" as used herein refers to an amount of a compound or a combination of compounds to treat or prevent a disease or disorder in a subject, or to treat a symptom of a disease or disorder. As used herein, the terms "subject" and "patient" generally refer to an individual who is undergoing or has undergone treatment (e.g., administration of a compound) according to a method described herein.

[0084] "Stable compound" and "stable structure" mean a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated by the present invention.

[0085] The terms "subject", "patient", or "subject in need thereof" refer to a living organism suffering from or susceptible to a disease or condition that can be treated by administration of a compound, pharmaceutical composition, mixture, or vaccine as provided herein. Non-limiting examples include humans, other mammals, bovine animals, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals. In some embodiments, the patient is a human. In some embodiments, the patient is a domesticated animal. In some embodiments, the patient is a dog. In some embodiments, the patient is a parrot. In some embodiments, the patient is a farm animal. In some embodiments, the patient is a mammal. In some embodiments, the patient is a cat. In some embodiments, the patient is a horse. In some embodiments, the patient is a bovine animal. In some embodiments, the patient is a canine animal. In some embodiments, the patient is a feline animal. In some embodiments, the patient is an ape. In some embodiments, the patient is a monkey. In some embodiments, the patient is a mouse. In some embodiments, the patient is a laboratory animal. In some embodiments, the patient is a rat. In some embodiments, the patient is a hamster. In some embodiments, the patient is a test animal. In some embodiments, the patient is a newborn animal. In some embodiments, the patient is a newborn human. In some embodiments the patient is a newborn mammal. In some embodiments the patient is an elderly animal. In some embodiments the patient is an elderly human. In some embodiments the patient is an elderly mammal. In some embodiments the patient is a geriatric patient.

[0086] The term "effective amount" as used herein refers to an amount effective to achieve the intended purpose. Thus, the term "therapeutically effective amount" and the like refers to the amount of a compound, mixture or vaccine, or a combination thereof, for treating or preventing a disease or disorder, or treating the symptoms of a disease or disorder, in a subject in need thereof.

[0087] The term "TLR" refers to the K This refers to Toll-like receptors, components of the innate immune system that regulate B activation.

[0088] The term "TLR modulator", "TLR immunomodulator" and the like as used herein refer to compounds that activate or inhibit Toll-like receptors in the usual and customary sense.See, for example, PCT / US2010 / 000369, Hennessy, EJ, et al., Nature Reviews 2010, 9:283- 307; PCT / US2008 / 001631; PCT / US2006 / 032371; PCT / US2011 / 000757.Thus, a "TLR agonist" is a TLR modulator that activates TLRs, and a "TLR antagonist" is a TLR modulator that inhibits TLRs.

[0089] The term "TLR4" as used herein refers to the TLR4 gene product, and homologs, isoforms, and functional fragments thereof: isoform 1 (NCBI accession NP_612564.1); isoform 2 (NCBI accession NP_003257.1); isoform 3 (NCBI accession NP_612567.1). Agonists of TLR4 that may be included in the disclosed formulations include, but are not limited to, compounds of formula (II), such as pyrimidoindoles, aminoalkyl glucosaminide phosphates, specifically CRX-601 and CRX-547), RC-29, monophosphoryl phospholipid A (MPL), glucopyranosyl lipid adjuvants (GLA and SLA), OM-174, PET lipid A, ONO-4007, INI-2004 (diamine allose phosphate), and E6020.

[0090] The term "TLR7" as used herein refers to the TLR7 gene product (NCBI accession AAZ99026), and homologs, and functional fragments thereof. TLR7 agonists that may be included in the disclosed formulations include, but are not limited to, compounds of formula (I), imidazoquinolines, such as imiquimod, CL097 or gardiquimide, adenine analogs such as CL264, CL087, thiazoloquinolines such as 3M002 (CL075), guanosine analogs such as asloxoribine, or thioquinolines.

[0091] [TLR4 and TLR7] Toll-like receptors (TLRs) are pattern recognition receptors that recognize conserved microbial products known as pathogen-associated molecular patterns (PAMPs). TLR4 recognizes LPS. TLR4 signaling activates MyD88 and TRIF-dependent pathways. The MyD88 pathway activates NF-κB and JNK to induce inflammatory responses. The TRIF pathway activates IRF3 to induce IFN-α production.

[0092] TLR4 is expressed primarily on monocytes, mature macrophages, as well as on dendritic cells, mast cells, and intestinal epithelium. TLR modulators (agonists) for TLR4 include NI-0101 (Hennessy 2010, Id.), 1A6 (Ungaro, R., et al., Am. J. Physiol. Gastrointest. Liver Physiol. 2009, 296:G1167-G1179), AV411 (Ledeboer, A., et al., Neuron Glia Biol. 2006, 2:279-291; Ledeboer, A., et al., Expert Opin. Investig. Drugs 2007, 16:935-950), Eritoran (Mullarkey, M., et al., J. Pharmacol. Exp. Ther. 2003, 305:1093-1102), and TAK-242 (Li, M., et al., J. Pharmacol. Exp. Ther. 2003, 305:1093-1102). al., Mol. Pharmacol. 2006, 69:1288-1295). TLR modulators (agonists) for TLR4 include Pollinex® Quattro (Baldrick, P., et al., J. Appl. Toxicol. 2007, 27:399-409; DuBuske, L., et al., J. Allergy Clin. Immunol. 2009, 123:S216). TLR7 signaling activates MyD88-dependent pathway and IRF7-dependent signaling. The IRF7 pathway induces IFN-α production.

[0093] TLR7 senses ss-RNA or synthetic chemicals (imiquimod, R848). TLR7 and TLR8 are found in the endosomes of monocytes and macrophages, TLR7 is also expressed in plasmacytoid dendritic cells, and TLR8 is also expressed in mast cells. Both of these receptors recognize single-stranded RNA from viruses. Synthetic ligands (e.g., R-848 and imiquimod) can be used to activate the signaling pathways of TLR7 and TLR8. See, for example, Caron, G., et al., J. Immunol. 2005, 175:1551-1557. TLR9 is expressed in the endosomes of monocytes, macrophages, and plasmacytoid dendritic cells and acts as a receptor for unmethylated CpG islands found in bacterial and viral DNA. Synthetic oligonucleotides containing unmethylated CpG motifs are used to activate TLR9. For example, class A oligonucleotides target plasmacytoid dendritic cells and strongly induce IFNa production and antigen-presenting cell maturation, while indirectly activating natural killer cells. Class B oligonucleotides target B cells and natural killer cells and barely induce interferon-a (IFNa). Class C oligonucleotides target plasmacytoid dendritic cells and are strong inducers of IFNa. This class of oligonucleotides is involved in the activation and maturation of antigen-presenting cells, indirectly activating natural killer cells, and directly stimulating B cells. For example, see Vollmer, J., et al., Eur. J. Immunol. 2004, 34:251-262; Strandskog, G., et al., Dev. Comp. Immunol. 2007, 31:39-51.

[0094] Reported TLR modulators (agonists) for TLR7 include ANA772 (Kronenberg, B. & Zeuzem, S., Ann. Hepatol. 2009, 8:103-112), imiquimod (Somani, N. & Rivers, JK, Skin Therapy Lett. 2005, 10:1-6), and AZD8848 (Hennessey 2010, Id.). TLR modulators (agonists) for TLR8 include VTX-1463 (Hennessey 2010, Id.). TLR modulators (agonists) for TLR7 and TLR8 include Resiquimod (Mark, KE, et al., J. Infect. Dis. 2007, 195:1324-1331; Pockros, PJ, et al., J. Hepatol. 2007, 47:174-182). TLR modulators (antagonists) for TLR7 and TLR9 include IRS-954 (Barrat, FJ, et al., Eur. J. Immunol. 2007, 37:3582-3586) and IMO-3100 (Jiang, W., et al., J. Immunol. 2009, 182:48.25).TLR9 agonists include SD-101 (Barry, M. & Cooper, C., Expert Opin. Biol. Ther. 2007, 7:1731-1737), IMO-2125 (Agrawal, S. & Kandimalla, ER, Biochem. Soc. Trans. 2007, 35:1461-1467), Bio Thrax plus CpG-7909 (Gu, M., et al., Vaccine 2007, 25:526-534), AVE0675 (Parkinson, T., Curr. Opin. Mol. Ther. 2008, 10:21-31), and QAX-935 (Panter, G., et al., Curr. Opin. Mol. Ther. 2009, 11:133-145), SAR-21609 (Parkinson 2008, Id.), and DIMS0150 (Pastorelli, L., et al., Expert Opin. Emerg. Drugs 2009, 14:505-521).

[0095] [TLR7 Ligands and Conjugates Thereof] As used herein with respect to TLR7 ligands and conjugates thereof, the terms "alkyl", "alkenyl" and "alkynyl" can include linear, branched and cyclic monovalent hydrocarbyl radicals, and combinations thereof, which, when unsubstituted, contain only C and H. For example, methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2 propenyl, 3 butynyl, etc. The total number of carbon atoms in each such group may be described herein. For example, 1-10C, or C1-C when the group can contain up to 10 carbon atoms. 10 Or C 1~10Where heteroatoms (typically N, O, and S) are allowed to substitute carbon atoms, such as in a heteroalkyl group, for example, the numbers describing the group are still written (e.g., C1-C6), but represent the total number of carbon atoms in the group and the number of such heteroatoms included as substitutions for carbon atoms in the backbone of the ring or chain being described.

[0096] Typically, the alkyl, alkenyl, and alkynyl substituents of the present invention contain one 10C (alkyl) or two 10C (alkenyl or alkynyl). For example, they contain one 8C (alkyl) or two 8C (alkenyl or alkynyl). Sometimes, they contain one 4C (alkyl) or two 4C (alkenyl or alkynyl). A single group may contain more than one type of multiple bond, or more than one multiple bond. Such groups are included within the definition of the term "alkenyl" if they contain at least one carbon-carbon double bond, and are included within the term "alkynyl" if they contain at least one carbon-carbon triple bond.

[0097] Alkyl, alkenyl, and alkynyl groups are often substituted to the extent that such substitution makes chemical sense. Representative substituents include, but are not limited to, halo, =O, =N-CN, =N-OR, =NR, OR, NR2, SR, SO2R, SO2NR2, NRS02R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, OOCR, COR, and NO2, where each R is independently selected from H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C8 heteroaryl ... 10 Aryl, or C5-C 10and each R is optionally substituted with halo, =O, =N-CN, =N-OR', =NR', OR', NR'2, SR', SOR', SO2NR'2, NR'SOR', NR'CONR'2, NR'COOR', NR'COR', CN, COOR', CONR'2, OOCR', COR', and NO2, where each R' is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C8 heteroaryl ... 10 Aryl, or C5-C 10 The alkyl, alkenyl, and alkynyl groups are also C1-C8 acyl, C2-C8 heteroacyl, C6-C8 heteroaryl, C7-C8 heteroaryl, C8-C9 heteroaryl, C9-C10 heteroaryl, C10-C11 heteroaryl, C11-C12 heteroaryl, C12-C13 heteroaryl, C13-C14 heteroaryl, C14-C15 heteroaryl, C15-C16 heteroaryl, C 10 Aryl, or C5-C 10 Heteroaryl can be substituted, each of which can be substituted by substituents suitable for the particular group.

[0098] The "acetylene" substituent may include an optionally substituted 2-10C alkynyl group and is represented by the formula -C≡C-Ri, where Ri is H or C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C7-C 12 Aryl alkyl or C6-C 12 Each R group is optionally substituted with one or more substituents selected from halo, =O, =N-CN, =N-OR', =NR', OR', NR'2, SR', SOR', SO2NR'2, NR'SOR', NR'CONR'2, NR'COOR', NR'COR', CN, COOR', CONR'2, OOCR', COR', and NO2, where each R' is independently selected from H, C1-C6 alkyl, C2-C6 heteroalkyl, C1-C6 acyl, C2-C6 heteroacyl, C6-C6 heteroaryl ... 10 Aryl, C5-C 10 Heteroaryl, C 7~12 Arylalkyl, or C6~12 Heteroarylalkyl, each of which is optionally substituted with one or more groups selected from halo, C1-C4 alkyl, C1-C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl, hydroxy, amino, and =O. Also, two R' may be joined to form a 3-7 membered ring, optionally containing up to three heteroatoms selected from N, O, and S. In some embodiments, Ri in -C≡C-Ri is H or Me.

[0099] "Heteroalkyl", "heteroalkenyl", and "heteroalkynyl", etc. are defined in the same manner as the corresponding hydrocarbyl (alkyl, alkenyl, and alkynyl) groups. However, the term "hetero" refers to a group containing 1-3 O, S, or N heteroatoms or combinations thereof in the backbone residue. Thus, at least one carbon atom of the corresponding alkyl, alkenyl, or alkynyl group is replaced by one of the specified heteroatoms to form a heteroalkyl, heteroalkenyl, or heteroalkynyl group. The typical size for the heterotypes of alkyl, alkenyl, and alkynyl groups is generally the same as the size of the corresponding hydrocarbyl group. The substituents that may be present on the heterotypes are the same as those described above for the hydrocarbyl groups. It is also to be understood that, unless otherwise stated, for reasons of chemical stability, such groups do not contain more than two consecutive heteroatoms, except when an oxo group is present on an N or S, such as in a nitro or sulfonyl group.

[0100] As used herein, "alkyl" includes cycloalkyl and cycloalkylalkyl groups, and the term "cycloalkyl" may be used herein to describe a carbocyclic non-aromatic group linked through a ring carbon atom, and "cycloalkylalkyl" may be used to describe a carbocyclic non-aromatic group linked to a molecule through an alkyl linker. Similarly, "heterocyclyl" may be used to describe a non-aromatic ring group that contains at least one heteroatom as a ring member and is linked to a molecule through a ring atom that may be C or N. "Heterocyclylalkyl" may be used to describe such a group that is linked to another molecule through a linker. The sizes and substituents suitable for cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl groups are the same as those described above for alkyl groups. As used herein, these terms also include rings that contain a double bond or two, unless the ring is aromatic.

[0101] As used herein, "acyl" includes groups containing an alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical attached to one of the two available valence positions of the carbonyl carbon atom. Heteroacyl refers to the corresponding group in which at least one carbon other than the carbonyl carbon is replaced with a heteroatom selected from N, O, and S. Heteroacyl includes, for example, -C(=O)OR and -C(=O)NR2, as well as -C(=O)-heteroaryl.

[0102] Acyl and heteroacyl groups are attached to any group or molecule to which they are attached through the open valence of the carbonyl carbon atom. Typically, they are C1-C8 acyl groups, including formyl, acetyl, pivaloyl, and benzoyl, and C2-C8 heteroacyl groups, including methoxyacetyl, ethoxycarbonyl, and 4-pyridinoyl. Hydrocarbyl groups, aryl groups, and heteroforms of such groups, including acyl or heteroacyl groups, can be substituted with the substituents described herein (generally appropriate for each of the components corresponding to the acyl or heteroacyl group).

[0103] An "aromatic" or "aryl" moiety refers to a monocyclic or fused bicyclic moiety having the well-known property of aromaticity, such as phenyl and naphthyl. Similarly, "heteroaromatic" and "heteroaryl" refer to a monocyclic or fused bicyclic system containing one or more heteroatoms selected from O, S, and N as ring members. The inclusion of heteroatoms allows for aromaticity in five-membered as well as six-membered rings. Typical heteroaromatic systems include monocyclic C5-C6 aromatic groups such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, and imidazolyl, as well as fused bicyclic moieties formed by fusing one of these monocyclic groups with either a phenyl ring or a heteroaromatic monocyclic group, and include C8-C6 aromatic groups such as indolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolyl, and the like. 10 Form a bicyclic group. Any monocyclic or fused bicyclic system that has the characteristic of aromaticity with respect to the electron distribution throughout the ring system is included in this definition. It also includes bicyclic groups in which at least the ring directly bonded to the rest of the molecule has the characteristic of aromaticity. Typically, the ring system contains 5 to 12 ring member atoms. For example, a monocyclic heteroaryl may contain 5 to 6 ring members, and a bicyclic heteroaryl contains 8 to 10 ring members.

[0104] The aryl and heteroaryl moieties are C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C 12 It may be substituted with a variety of substituents including aryl, C1-C8 acyl, and hetero forms thereof, each of which may itself be further substituted. Alternative substituents for the aryl and heteroaryl moieties include halo, OR, NR2, SR, SO2R, SO2NR2, NRS02R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, OOCR, COR, and NO2, where each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C8 heteroaryl, C8-C8 heteroaryl ... 10 Aryl, C5-C 10 Heteroaryl, C7-C 12 Aryl alkyl or C6-C 12 Heteroarylalkyl, each R is optionally substituted as described above for alkyl groups. Substituents on aryl or heteroaryl groups can of course be further substituted with groups as described herein, as appropriate for each type of such substituent or each component of the substituent.Thus, for example, arylalkyl substituents can be substituted in the aryl portion with the substituents described herein, as typical for aryl groups, and can be further substituted in the alkyl portion with the substituents described herein, as typical for or appropriate for alkyl groups.

[0105] Similarly, "arylalkyl" and "heteroarylalkyl" refer to aromatic and heteroaromatic ring systems that are connected to their points of attachment via linking groups, such as alkylene, including substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic linkers. Typically, the linkers are C1-C8 alkyls or heteroforms thereof. These linkers may also contain carbonyl groups, so that they can provide substituents as acyl or heteroacyl moieties. The aryl or heteroaryl rings in the arylalkyl or heteroarylalkyl groups may be substituted with the same substituents as described above for the aryl group. For example, arylalkyl groups include phenyl rings (optionally substituted with groups defined above for the aryl group) and C1-C4 alkylenes (unsubstituted or substituted with one or two C1-C4 alkyl or heteroalkyl groups, where the alkyl or heteroalkyl groups may be optionally cyclized to form rings such as cyclopropane, dioxolane, or oxacyclopentane). Similarly, heteroarylalkyl groups include C5-C6 monocyclic heteroaryl groups (optionally substituted with groups listed above as typical substituents for aryl groups) and C1-C4 alkylene (unsubstituted or substituted with one or two C1-C4 alkyl or heteroalkyl groups), or optionally substituted phenyl rings, or C5-C6 monocyclic heteroaryl and C1-C4 heteroalkylene (unsubstituted or substituted with one or two C1-C4 alkyl or heteroalkyl groups, where the alkyl or heteroalkyl groups may be optionally cyclized to form a ring such as cyclopropane, dioxolane, or oxacyclopentane).

[0106] When an arylalkyl or heteroarylalkyl group is described as being optionally substituted, the substituents may be on either the alkyl or heteroalkyl portion of the group, or on the aryl or heteroaryl portion of the group. The optional substituents on the alkyl or heteroalkyl portion are generally the same as those described above for an alkyl group, and the optional substituents on the aryl or heteroaryl portion are generally the same as those described above for an aryl group.

[0107] As used herein, "arylalkyl" groups are hydrocarbyl groups when they are unsubstituted and are described by the total number of carbon atoms in the ring and the alkylene, or similar linker. Thus, a benzyl group is a C7-arylalkyl group and a phenylethyl is a C8-arylalkyl group.

[0108] The above "heteroarylalkyl" refers to a moiety containing an aryl group bonded through a linking group, and differs from "arylalkyl" in that at least one ring atom of the aryl moiety or one atom of the linking group is a heteroatom selected from N, O, and S. Heteroarylalkyl groups are described herein according to the total number of atoms of the ring and the linker to which they are attached, and include aryl groups bonded through a heteroalkyl linker; heteroaryl groups bonded through a hydrocarbyl linker such as alkylene; and heteroaryl groups bonded through a heteroalkyl linker. Thus, for example, C7-heteroarylalkyl includes pyridylmethyl, phenoxy, and N-pyrrolylmethoxy.

[0109] As used herein, "alkylene" refers to a divalent hydrocarbyl group, which, because it is divalent, may link two other groups together. Typically, -(CH2) n-, where n is 1-8, for example, n is 1-4, but where specified, the alkylene may be substituted with other groups, may be of other lengths, and the open valences need not be at both ends of the chain. Thus, -CH(Me)- and -C(Me)2- are also referred to as alkylenes, as are cyclic groups such as cyclopropane-1,1-diyl. When an alkylene group is substituted, the substituents include those typically present in alkyl groups as described herein.

[0110] In general, any alkyl, alkenyl, alkynyl, acyl, or aryl or arylalkyl group contained in a substituent, or any heteroform of one of these groups, may itself be optionally substituted by additional substituents. The nature of these substituents is the same as that recited for the original substituent itself if the substituent is not otherwise described. Thus, for example, R 2 When the embodiment of is alkyl, the alkyl is selected from the group consisting of R 2 It may be optionally substituted by the remaining substituents listed as embodiments of the alkyl group, without violating the size limitations provided for the alkyl group itself; for example, an alkyl group substituted by an alkyl or an alkenyl group merely extends the upper carbon atom limit of these embodiments, and does not include the alkyl group. However, alkyl groups substituted by aryl, amino, alkoxy, =O, etc. are included within the scope of the present invention, and the atoms of these substituents are not counted in the number used to describe the alkyl, alkenyl, etc. group described. When the number of substituents is not specified, each of such alkyl, alkenyl, alkynyl, acyl, or aryl groups may be substituted with several substituents according to its available valences; in particular, for example, any of these groups may be substituted with a fluorine atom at any or all of its available valences.

[0111] In various embodiments, the present invention provides a method for preventing, inhibiting, or treating liver disease, such as that associated with inflammation, in a mammal, comprising administering to a mammal in need thereof an effective amount of a compound of formula (I):

[0112] [ka]

[0113] where X 1 is -O-, -S-, or -NR c - and; R 1 is hydrogen, (C1-C 10 ) Alkyl, Substituted (C1-C 10 ) Alkyl, C 6~10 Aryl or substituted C 6~10 Aryl, C 5~9 Heterocyclic, substituted C 5~9 is heterocyclic; R c is hydrogen, C 1~10 Alkyl or substituted C 1~10 alkyl; or R c and R 1 forms, together with the nitrogen to which it is attached, a heterocyclic ring or a substituted heterocyclic ring; Each R 2 are independently -OH, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 )Aryl (aroyl), substituted -C(O)-(C6-C 10 )Aryl, -C(O)OH (carboxyl), -C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -NR a R b , -C(O)NR a R b (carbamoyl), halo, nitro, or cyano, or R 2 is lacking; Each R a and R b are independently hydrogen, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het(C1-C6)alkyl, or (C1-C6)alkoxycarbonyl; where the substituents on any alkyl, aryl, or heterocyclic group are hydroxy, C 1~6 Alkyl, Hydroxy C 1~6 Alkylene, C 1~6 Alkoxy, C 3~6 Cycloalkyl, C 1~6 Alkoxy C 1~6 alkylene, amino, cyano, halo, or aryl; n is 0, 1, 2, 3, or 4; X 2 is a bond or a linking group; and In one embodiment, R X is a phospholipid containing one or two carboxylate esters, or -(R 3 ) r -(R 4 ) s ) p Including R 3 are independently polyethylene glycol (PEG) moieties, R 4 are independently H, -C1-C6 alkyl, -C1-C6 alkoxy, -NR a R b , -N3, -OH, -CN, -COOH, -COOR 1 , -C1~C6 alkyl-NR a R b , C1~C6 alkyl-OH, C1~C6 alkyl-CN, C1~C6 alkyl-COOH, C1~C6 alkyl-COOR 1, a 5-6 membered ring, a substituted 5-6 membered ring, -C1-C6 alkyl-5-6 membered ring, -C1-C6 alkyl-substituted 5-6 membered ring, a C2-C9 heterocyclic ring, or a substituted C2-C9 heterocyclic ring, where r is 1-1000, s is 1-100, and p is 1-100; or a tautomer thereof; or a pharma- ceutically acceptable salt or solvate thereof.

[0114] In one embodiment, R 3 is a PEG moiety.

[0115] In some embodiments, the PEG reactant has the structure CH3O(CH2CH2O)nX-NHS * In certain embodiments, the PEG reactant has the following structure:

[0116] [ka]

[0117] has.

[0118] In some embodiments, certain PEG reactants are bifunctional. In some embodiments, examples of bifunctional PEG reactants have the structure X-(OCH2CH2)nX, where X is (N-succinimidyloxycarbonyl)methyl (-CH2COO-NHS), succinimidyl glutarate (-COCH2CH2CH2COO-NHS), (N-succinimidyloxycarbonyl)pentyl (-(CH2)5COO-NHS), 3-(n-maleimidyl)propanamide, (-NHCOCH2CH2-MAL), aminopropyl (-CH2CH2CH2NH2), or 2-sulfanylethyl (-CH2CH2SH).

[0119] In certain embodiments, some PEG reactants are heterofunctional. Examples of heterofunctional PEG reactants include those with the following structures:

[0120] [ka]

[0121] where, in some embodiments, X can be N-succinimidyloxycarbonyl)methyl (-CHCOO-NHS), succinimidyl glutarate (-COCHCHCHCOO-NHS), (N-succinimidyloxycarbonyl)pentyl (-(CH)COO-NHS), 3-(n-maleimidyl)propanamide, (-NHCOCHCH-MAL), 3-aminopropyl (-CHCHCHNH), 2-sulfanylethyl (-CHCHSH), 5-(N-succinimidyloxycarbonyl)pentyl (-(CH)COO-NHS), or p-nitrophenyloxycarbonyl, (-CO-p-CHNO).

[0122] Certain branched PEG reactants may also be utilized, such as those having the following structures:

[0123] [ka]

[0124] where X is a spacer and Y is a functional group, which in some embodiments includes, but is not limited to, maleimide, amine, glutaryl-NHS, carbonate-NHS, or carbonate-p-nitrophenol. An advantage of branched PEG reactants is that they can yield conjugated products with sustained release properties.

[0125] In certain embodiments, the PEG reactant may also be a heterofunctional reactant, such as:

[0126] [ka]

[0127] In some embodiments, Boc * -protected-amino-PEG-carboxyl-NHS or maleimide-PEG-carboxyl-NHS reactants may be utilized.

[0128] In certain embodiments, comb polymers can be utilized as PEG reactants to incorporate several PEG units into the conjugate. An example of a comb polymer is shown below.

[0129] [ka]

[0130] In some embodiments, the PEG reactant and / or PEG conjugate product may have a molecular weight ranging from about 5 grams per mole to about 100,000 grams per mole. In some embodiments, the PEG reactant and / or PEG conjugate product has an average, mean, or nominal molecular weight of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, or 90000 grams per mole. In some embodiments, the PEG moieties in the compounds herein are uniform, that is, the molecular weight of the PEG moiety is the same for each molecule of a particular group of compounds (e.g., R 3 is one PEG unit and r is 2-10).

[0131] In various embodiments, X in formula (I) 2can be a bond or a chain having 1 to about 10 atoms in the chain, where the atoms of the chain are selected from the group consisting of carbon, nitrogen, sulfur, and oxygen, where any carbon atom can be substituted with oxo and any sulfur atom can be substituted with one or two oxo groups. The chain can be interspersed with one or more cycloalkyl, aryl, heterocyclyl, or heteroaryl rings.

[0132] X in formula (I) 2 Specific examples of -(Y) y -, -(Y) y -C(O)N-(Z) z -, -(CH2) y -C(O)N-(CH2) z -, -(Y) y -NC(O)-(Z) z -, -(CH2) y -NC(O)-(CH2) z In this case, each y (index) and z (index) is independently 0 to 20, and each Y and Z is independently C1 to C 10 Alkyl, substituted C1-C 10 Alkyl, C1-C 10 Alkoxy, substituted C1-C 10 Alkoxy, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, C5-C 10 Aryl, substituted C5-C 10 Aryl, C5-C9 heterocyclic, substituted C5-C9 heterocyclic, C1-C6 alkanoyl, Het, HetC1-C6 alkyl, or C1-C6 alkoxycarbonyl, where the substituents on the alkyl, cycloalkyl, alkanoyl, alkoxycarbonyl, Het, aryl, or heterocyclic groups are hydroxyl, C1-C 10 Alkyl, hydroxyl C1-C 10 Alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9 heterocyclic, C 1~6 Alkoxy C 1~6alkenyl, amino, cyano, halogen, or aryl. In certain embodiments, the linker can be a -C(Y')(Z')-C(Y'')(Z'')- linker, where each Y', Y'', Z', and Z'' is independently a hydrogen C1-C 10 Alkyl, substituted C1-C 10 Alkyl, C1-C 10 Alkoxy, substituted C1-C 10 Alkoxy, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, C5-C 10 Aryl, substituted C5-C 10 Aryl, C5-C9 heterocyclic, substituted C5-C9 heterocyclic, C1-C6 alkanoyl, Het, HetC1-C6 alkyl, or C1-C6 alkoxycarbonyl, where the substituents on the alkyl, cycloalkyl, alkanoyl, alkoxycarbonyl, Het, aryl, or heterocyclic groups are hydroxyl, C1-C 10 Alkyl, hydroxyl C1-C 10 Alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9 heterocyclic, C1 ~ 6AlkoxyC 1~6 It is alkenyl, amino, cyano, halogen, or aryl.

[0133] X in formula (I) 2 Another specific value of is

[0134] [ka]

[0135] It is.

[0136] X 2 Another specific value of is

[0137] [ka]

[0138] It is.

[0139] In various embodiments, X 2 is either C(O) or

[0140] [ka]

[0141] It could be.

[0142] In various embodiments, X in formula (I) 1 may be oxygen.

[0143] In various embodiments, X in formula (I) 1 -Sulfur or -NR c -, where R c is hydrogen, C 1~6 Alkyl or substituted C 1~6 Alkyl, the alkyl substituents are hydroxy, C 3~6 Cycloalkyl, C 1~6 alkoxy, amino, cyano, or aryl. More specifically, X 1 can be -NH-.

[0144] In various embodiments, R in formula (I) 1 and R c may be taken together to form a heterocyclic or substituted heterocyclic ring. More specifically, R 1 and R c may be taken together to form a substituted or unsubstituted morpholino, piperidino, pyrrolidino, or piperazino ring.

[0145] In various embodiments, R in formula (I) 1 is C 1~6 Alkoxy-substituted C1-C 10 It may be alkyl.

[0146] In various embodiments, R in formula (I) 1 is hydrogen, C 1~4Alkyl or substituted C 1-4 More specifically, R 1 is hydrogen, methyl, ethyl, propyl, butyl, hydroxyC 1~4 Alkylene, or C 1~4 Alkoxy C 1~4 Even more specifically, R 1 can be hydrogen, methyl, ethyl, methoxyethyl, or ethoxyethyl.

[0147] In various embodiments, R in formula (I) 2 may be absent, or R 2 is halogen or C 1~4 More specifically, R 2 can be chloro, bromo, methyl, or ethyl.

[0148] In one embodiment, R in formula (I) x is ((R 3 ) r -(R 4 ) s ) p , or R 3 In one embodiment, R 3 is a PEG moiety or a derivative of a PEG moiety. In certain embodiments, R 3is -O-CH2-CH2- or -CH2-CH2-O-. In one embodiment, the PEG moiety can include one or more PEG units. The PEG moiety can include from about 1 to about 1,000 PEG units, in some embodiments, including but not limited to about 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, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 units. In certain embodiments, the PEG moiety may comprise about 1 to 5 up to about 25 PEG units, about 1 to 5 up to about 10 PEG units, about 10 up to about 50 PEG units, about 18 up to about 50 PEG units, about 47 up to about 150 PEG units, about 114 up to about 350 PEG units, about 271 up to about 550 PEG units, about 472 up to about 950 PEG units, about 50 up to about 150 PEG units, about 120 up to about 350 PEG units, about 250 up to about 550 PEG units, or about 650 up to about 950 PEG units. In certain embodiments, the PEG unit is -O-CH2-CH2- or -CH2-CH2-O-. In some embodiments, R 4 is H, -C1-C6 alkyl, -C1-C6 alkoxy, -NR a R b , -N3, -OH, -CN, -COOH, -COOR 1 , -C1~C6 alkyl-NR a R b , C1~C6 alkyl-OH, C1~C6 alkyl-CN, C1~C6 alkyl-COOH, C1~C6 alkyl-COOR 1 , a 5- or 6-membered ring, a substituted 5- or 6-membered ring, -C1-C6 alkyl-5- or 6-membered ring, -C1-C6 alkyl-substituted 5- or 6-membered ring C2-C9 heterocyclic ring, or a substituted C2-C9 heterocyclic ring.

[0149] In some embodiments, r is from about 5 to about 100, and sometimes r is from about 5 to about 50 or from about 5 to about 25. In certain embodiments, r is from about 5 to about 15, and sometimes r is about 10. In some embodiments, R 3 is a PEG unit (PEG) r and r is from about 2 to about 10 (for example, r is from about 2 to about 4) or from about 18 to about 500.

[0150] In some embodiments, s is from about 5 to about 100, and sometimes s is from about 5 to about 50 or from about 5 to about 25. In certain embodiments, s is from about 5 to about 15, and sometimes s is about 10. In some embodiments, s is about 5 or less (e.g., s is 1). In some embodiments, (R 3 ) r The substituents are linear and in certain embodiments, (R 3 ) r The substituent is branched. In the case of linear moieties, s may be less than r (e.g., when R3 is -O-CH2-CH2- or -CH2-CH2-O-), and sometimes s is 1. In some embodiments, R3 is a linear PEG moiety (e.g., having about 1 to about 1000 PEG units), s is 1, and r is 1. In the case of branched moieties, s may be less than, greater than, or equal to r (e.g., when R3 is -O-CH2-CH2- or -CH2-CH2-O-). 3 is -O-CH2-CH2- or -CH2-CH2-O-), and sometimes r is 1, s is 1 and p is from about 1 to about 1000 (e.g., p is about 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, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000).

[0151] In some embodiments, R 3is -O-CH2-CH2- or -CH2-CH2-O-, and r is about 1 to about 1000 (e.g., about 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, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000).

[0152] In certain embodiments, X 2 is an amide linking group (e.g., -C(O)NH- or -NH(O)C-); an alkylamide linking group (e.g., -C1-C6 alkyl-C(O)NH-, -C1-C6 alkyl-NH(O)C-, -C(O)NH-C1-C6 alkyl-, -NH(O)C-C1-C6 alkyl-, -C1-C6 alkyl-NH(O)C-C1-C6 alkyl-, -C1-C6 alkyl-NH(O)C-C1-C6 alkyl-, -C1-C6 alkyl-C(O)NH-C1-C6 alkyl-, or -C(O)NH-(CH2) t -, where t is 1, 2, 3, or 4; a substituted 5- to 6-membered ring (e.g., an aryl ring, a heteroaryl ring (e.g., tetrazole, pyridyl, 2,5-pyrrolidinedione (e.g., 2,5-pyrrolidinedione substituted with a substituted phenyl moiety)), a carbocyclic ring, or a heterocyclic ring), or an oxygen-containing moiety (e.g., -O-, -C1-C6 alkoxy).

[0153] As used herein, the term "phospholipid" refers to a glycerol mono- or diester having a phosphate group attached to the glycerol hydroxyl group, and having an alkanolamine group attached as an ester to the phosphate group, represented by the general formula:

[0154] [ka]

[0155] Here, R 11 and R 12 are each independently hydrogen or an acyl group; R 13 R is either a negative charge or a hydrogen, depending on the pH.13 If m is negatively charged, an appropriate counterion such as a sodium ion may be present. For example, the alkanolamine moiety may be an ethanolamine moiety such that m=1. It should also be understood that the NH group may be protonated and positively charged, or unprotonated and neutral, depending on the pH. For example, phospholipids may exist as zwitterions with a negatively charged phosphate oxyanion and a positively charged protonated nitrogen atom. OR 12 Since the carbon atom bearing the R atom is a chiral carbon atom, the molecule may exist as the R isomer, the S isomer, or any mixture thereof. When there are equal amounts of R and S isomers in a sample of a compound of formula (II), the sample is called a "racemic mixture." For example, in the commercially available product 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, the R 3 The group has a chiral structure

[0156] [ka]

[0157] which is of the R absolute configuration.

[0158] The phospholipids can be free molecules or can be covalently attached to another group, for example as shown below:

[0159] [ka]

[0160] Here, the wavy lines indicate the attachment points.

[0161] Thus, R of the compound of formula (I) herein xWhen a substituent such as is described as being a phospholipid, this means that the phospholipid group is attached to another group, such as an N-benzyl heterocyclic system as disclosed herein, as specified by its structure. The point of attachment of the phospholipid group may be at any chemically feasible location, such as by structural depiction, unless otherwise specified. For example, in the above phospholipid structure, the point of attachment to another chemical moiety may be at the ethanolamine nitrogen atom (e.g., an amide group by bonding to a carbonyl group of another chemical moiety (e.g.,

[0162] [ka]

[0163] where R represents another chemical moiety to which the phospholipid is attached. In this attached amide derivative, R 13 The group may be a proton or a negative charge associated with a counterion such as a sodium ion. The acylated nitrogen atom of the alkanolamine group is no longer a basic amine but an electrically neutral amide, which itself is not protonated at physiological pH.

[0164] An "acyl" group, as that term is used herein, refers to an organic structure having a carbonyl group through which the structure is attached to, for example, a glycerol hydroxyl group of a phospholipid to form a "carboxylic acid ester" group. Examples of acyl groups include fatty acid groups, such as an oleoyl group, thus forming a fatty (e.g., oleoyl) ester with the glycerol hydroxyl group. Thus, R 11 or R 12 When one (but not both) of R is an acyl group, the phospholipid is a mono-carboxylate ester, and R 11 and R 12 When both are acyl groups, the phospholipid is a di-carboxylate ester.

[0165] In one embodiment, R xThe phospholipids of contain two carboxylate esters, each of which contains one, two, three, or four sites of unsaturation, epoxidation, hydroxylation, or a combination thereof.

[0166] In one embodiment, R x The phospholipids of the present invention contain two carboxylate esters, which may be the same or different.

[0167] In one embodiment, each carboxylic acid ester of the phospholipid is a C17 carboxylic acid ester having an unsaturated site at C8-C9.

[0168] In one embodiment, each carboxylic acid ester of the phospholipid is a C18 carboxylic acid ester having an unsaturated site at C9-C10.

[0169] In one embodiment, X 2 is a bond or a chain having 1 to about 10 atoms in the chain, the atoms in the chain being selected from the group consisting of carbon, nitrogen, sulfur, and oxygen (any carbon atom can be substituted with oxo and any sulfur atom can be substituted with one or two oxo groups).

[0170] In one embodiment, X 2 is C(O),

[0171] [ka]

[0172] It is.

[0173] In one embodiment, R x contains dioleoylphosphatidylethanolamine (DOPE).

[0174] In one embodiment, R x is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and X 2 is C(O).

[0175] In one embodiment, X 1 is oxygen or -NH-.

[0176] In one embodiment, R 1 and R c taken together form a heterocyclic or substituted heterocyclic ring, for example a substituted or unsubstituted morpholino, piperidino, pyrrolidino, or piperazino ring.

[0177] In one embodiment, R 1 is C1-C10 alkyl substituted with C1-6 alkoxy, R 1 is hydrogen, C 1~4 Alkyl or substituted C 1~4 Alkyl or R 1 is hydrogen, methyl, ethyl, propyl, butyl, hydroxyC 1~4 Alkylene, or C 1~4 Alkoxy C 1~4 alkylene or R 1 is hydrogen, methyl, ethyl, methoxyethyl, or ethoxyethyl.

[0178] In one embodiment, the composition further comprises an amount of an antigen.

[0179] In various embodiments, the mammal may be a human.

[0180] In various embodiments, the compositions may be administered intranasally, transdermally, or systemically.

[0181] [TLR4 Ligand] As used herein with respect to TLR4 ligands, the term "alkyl," alone or as part of another substituent, means, unless otherwise stated, straight-chain (i.e., unbranched) or branched-chain, or combinations thereof, which may be fully saturated, monounsaturated, or polyunsaturated, and have the specified number of carbon atoms (i.e., C1-C6). 10means 1-10 carbons) (can include divalent and polyvalent radicals). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, and homologs and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl groups are those with one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. Alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-).

[0182] The term "alkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from alkyl, including, but not limited to, -CH2CH2CH2CH2-. Typically, alkyl (or alkylene) groups have from 1 to 24 carbon atoms. In one embodiment, these groups have 10 or fewer carbon atoms. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms.

[0183] The term "heteroalkyl," alone or in combination with other terms, means, unless otherwise specified, a stable linear or branched chain, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, where the nitrogen and sulfur atoms are optionally oxygenated and the nitrogen heteroatom is optionally tetravalent. The heteroatoms O, N, P, S, and Si can be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3.

[0184] The term "heteroalkylene," alone or as part of another substituent, means, unless otherwise stated, a divalent radical derived from a heteroalkyl, including, but not limited to, -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). It is further noted that for alkylene and heteroalkylene linking groups, the direction in which the formula of the linking group is written does not imply an orientation of the linking group. For example, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-. As noted above, heteroalkyl groups as used herein include groups that are attached to the remainder of the molecule via a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SOR'. When reciting "heteroalkyl" followed by a recitation of a specific heteroalkyl group, such as -NR'R'', it will be understood that the terms heteroalkyl and -NR'R'' are not overlapping or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, as used herein, the term "heteroalkyl" should not be construed to exclude specific heteroalkyl groups, such as -NR'R''.

[0185] The terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, refer to cyclic versions of "alkyl" and "heteroalkyl", respectively, unless otherwise stated. Additionally, for heterocycloalkyl, a heteroatom may occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a "heterocycloalkylene," alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0186] The terms "halo" or "halogen," alone or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0187] The term "acyl," unless otherwise noted, means -C(O)R, where R is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0188] The term "aryl" refers to a polyunsaturated, aromatic, hydrocarbon substituent that may be a single ring or multiple rings (e.g., 1-3 rings, fused together (i.e., fused ring aryl) or covalently linked), unless otherwise stated. Fused ring aryl refers to 15 multiple rings fused together, where at least one of the fused rings is an aryl ring. The term "heteroaryl" refers to an aryl group (or ring) that contains at least one heteroatom selected from N, O, and S, where the nitrogen and sulfur atoms are optionally oxygenated and the nitrogen atom is optionally tetravalent. Thus, the term "heteroaryl" includes fused ring heteroaryl groups (i.e., multiple rings fused together and at least one of the fused rings is a heteroaromatic ring). 5,6-fused ring heteroarylene refers to two rings fused together, where one ring has 5 members and the other ring has 6 members, and at least one ring is a heteroaryl ring. Similarly, a 6,6-fused ring heteroarylene refers to two rings fused together, where one ring has 6 members and the other ring has 6 members, and at least one ring is a heteroaryl ring. Also, a 6,5-fused ring heteroarylene refers to two rings fused together, where one ring has 6 members and the other ring has 5 members, and at least one ring is a heteroaryl ring. The heteroaryl group may be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, Examples include 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene" alone or as part of another substituent mean a divalent radical derived from an aryl and heteroaryl, respectively.

[0189] For brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), and to include those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced, for example, by an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.).

[0190] The term "oxo," as used herein, means an oxygen that is double bonded to a carbon atom.

[0191] The term "alkylsulfonyl," as used herein, refers to a moiety having the formula -S(O2)-R', where R' is an alkyl group as defined above. R' may have a specified number of carbons (e.g., "C1-C4 alkylsulfonyl").

[0192] Each of the above terms (eg, "alkyl," "heteroalkyl," "aryl" and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical.

[0193] Substituents for an alkyl or heteroalkyl radical (including alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups) may be any of the following radicals in a number ranging from 0 to (2m'+1), where m' is the total number of carbon atoms in the radical: -OR', =0, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -O It can be one or more of a variety of groups selected from, but not limited to, C(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R'')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -CN, and -NO2. In one embodiment, R', R'', R''', and R'''' each independently refer to a hydrogen group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocycloalkyl group, a substituted or unsubstituted aryl group (e.g., an aryl group substituted with 1-3 halogens), a substituted or unsubstituted alkyl group, an alkoxy group, or a thioalkoxy group, or an arylalkyl group. When a compound of the invention includes more than one R group, e.g., when more than one group is present, each R group is independently selected as an R', R'', R''', and R'''' group, respectively. When R' and R'' are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups that have carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF and -CHCF) and acyls (e.g., -C(O)CH, -C(O)CF, -C(O)CHOCH, etc.).

[0194] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups can be varied and include, for example, -OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', - In one embodiment, R', R'', R''', and R'' are independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one R group, e.g., when more than one group is present, each R group is independently selected as an R', R'', R''', and R'''' group, respectively.

[0195] Two or more substituents may optionally form an aryl group, a heteroaryl group, a cycloalkyl group, or a heterocycloalkyl group. Such so-called ring-forming substituents are typically, but not necessarily, found attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of the cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of the cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.

[0196] Two substituents at adjacent atoms of an aryl or heteroaryl ring may optionally be represented by the formula -TC(O)-(CRR') q Alternatively, two substituents at adjacent atoms of an aryl or heteroaryl ring may optionally form a ring of the formula -U-, where T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer from 0 to 3. Alternatively, two substituents at adjacent atoms of an aryl or heteroaryl ring may optionally form a ring of the formula -A-(CH2) r Optionally, one of the single bonds of the new ring so formed may be replaced by a substituent of the formula -(CRR') s -X'(C''R''') d - (wherein s and d are independently integers from 0 to 3 and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-). In one embodiment, the substituents R, R', R'', and R''' are independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0197] As used herein, the term "heteroatom" or "ring heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0198] "Substituent," as used herein, means a group selected from the following moieties: (A) -OH, -NH2, -SH, -CN, -CF3, -CCl3, -NO2, oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, which are substituted with at least one group selected from the following: (i) oxo, -OH, -NH2, -SH, -CN, -CF3, -CCl3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, which are substituted with at least one group selected from the following: (a) oxo, -OH, -NH2, -SH, -CN, -CF3, -CCl3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, which are substituted with at least one group selected from the following: oxo, -OH, -NH2, -SH, -CN, -CF3, -CCl3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

[0199] A "size-limited substituent" or "size-limited substituent group" as used herein means a group selected from all of the substituents listed above for "substituent" (wherein each of the substituted or unsubstituted alkyl groups is a substituted or unsubstituted C1-C 20 each of the substituted or unsubstituted heteroalkyls is a substituted or unsubstituted 2-20 membered heteroalkyl, each of the substituted or unsubstituted cycloalkyls is a substituted or unsubstituted C4-C8 cycloalkyl, and each of the substituted or unsubstituted heterocycloalkyls is a substituted or unsubstituted 4-8 membered heterocycloalkyl).

[0200] A "lower substituent" or "lower substituent group", as used herein, means a group selected from all of the substituents described above for "substituent" (where, for example, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5-C7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5-7 membered heterocycloalkyl). In some embodiments, each of the substituents described in the compounds herein is substituted with at least one substituent.More specifically, in some embodiments, each of the substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein is substituted with at least one substituent.In other embodiments, at least one or all of these groups are substituted with at least one size-limiting substituent.In other embodiments, at least one or all of these groups are substituted with at least one lower-ranking substituent.

[0201] In some embodiments, the compounds described herein include a number of exemplary substituents, such as R 5 , R 5A , R 5B , R 5C , R 6A , R 6B , R 6C , R 7 , R 7A , R 7B , R 7C , R 8 , R 8A , R 8B , and / or R 8CIn such embodiments, each substituent may optionally be different at each occurrence and may be appropriately labeled to distinguish and better define each group. For example, each R 5A are different, they are, for example, R 5A.1 , R 5A.2 , R 5A.3 , R 5A.4 , R 5A.5 Similarly, R 5A , R 5B , R 5C , R 6A , R 6B , R 6C , R 7 , R 7A , R 7B , R 7C , R 8 , R 8A , R 8B , and / or R 8C If any of the following is present in combination, R 5A , R 5B , R 5C , R 6A , R 6B , R 6C , R 7 , R 7A , R 7B , R 7C , R 8 , R 8A , R 8B , and / or R 8C The definition of each existence is R 5A , R 5B , R 5C , R 6A , R 6B , R 6C , R 7 , R 7A , R 7B , R 7C , R 8 , R 8A , R 8B , and / or R 8C Assume the definition of

[0202] In one embodiment, the compound having the formula (II):

[0203] [ka]

[0204] or a pharma- ceutically acceptable salt thereof. In formula (II), z1 is an integer of 0 to 4, z2 is an integer of 0 to 5, and R 5 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 6 is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 7 is hydrogen or substituted or unsubstituted alkyl; R 8 is independently halogen, -CN, -SH, -OH, -COOH, -NH2, -CONH2, nitro, -CF3, -CCl3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0205] In one embodiment, R 5 is R 5A -substituted or unsubstituted cycloalkyl, R 5A Substituted or unsubstituted heterocycloalkyl, R 5A Substituted or unsubstituted aryl, or R 5A R is a substituted or unsubstituted heteroaryl. 5A are independently halogen, -CN, -CF3, -CCl3, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, R 5B -substituted or unsubstituted alkyl, R 5B -substituted or unsubstituted heteroalkyl, R 5B -substituted or unsubstituted cycloalkyl, R 5B -substituted or unsubstituted heterocycloalkyl, R 5B-substituted or unsubstituted aryl, or R 5B -substituted or unsubstituted heteroaryl. 5B are independently halogen, -CN, -CF3, -CCl3, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, R 5C -substituted or unsubstituted alkyl, R 5C -substituted or unsubstituted heteroalkyl, R 5C -substituted or unsubstituted cycloalkyl, R 5C -substituted or unsubstituted heterocycloalkyl, R 5C -substituted or unsubstituted aryl, or R 5C -substituted or unsubstituted heteroaryl. 5C is independently halogen, -CN, -CF3, -CCl3, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

[0206] Furthermore, according to this embodiment, R 6 is R 6A -substituted or unsubstituted alkyl, R 6A Substituted or unsubstituted heteroalkyl, R 6A Substituted or unsubstituted cycloalkyl, R 6A Substituted or unsubstituted heterocycloalkyl, R 6A Substituted or unsubstituted aryl, or R 6A R is a substituted or unsubstituted heteroaryl. 6A are independently halogen, -CN, -CF3, -CCl3, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, R 6B -substituted or unsubstituted alkyl, R 6B -substituted or unsubstituted heteroalkyl, R 6B -substituted or unsubstituted cycloalkyl, R 6B -substituted or unsubstituted heterocycloalkyl, R 6B -substituted or unsubstituted aryl, or 10R 6B-substituted or unsubstituted heteroaryl. 6B are independently halogen, -CN, -CF3, -CCl3, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, R 6C -substituted or unsubstituted alkyl, R 6C -substituted or unsubstituted heteroalkyl, R 6C -substituted or unsubstituted cycloalkyl, R 6C -substituted or unsubstituted heterocycloalkyl, R 6C -substituted or unsubstituted aryl, or R 6C -substituted or unsubstituted heteroaryl. 6C is independently halogen, -CN, -CF3, -CCl3, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

[0207] Furthermore, according to this embodiment, R 7 is hydrogen or R 7A -substituted or unsubstituted alkyl. 7A is independently halogen, -CN, -CF3, -CCl3, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

[0208] In addition to this embodiment, R 8 are independently halogen, -CN, -SH, -OH, -COOH, -NH2, -CONH2, nitro, -CF3, -CCl3, R 8A -substituted or unsubstituted alkyl, R 8A -substituted or unsubstituted heteroalkyl, R 8A Substituted or unsubstituted cycloalkyl, R 8A -substituted or unsubstituted heterocycloalkyl, R 8A Substituted or unsubstituted aryl, or R 8A-substituted or unsubstituted heteroaryl. 8A are independently halogen, -CN, -CF3, -CCl3, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, R 8B -substituted or unsubstituted alkyl, R 8B -substituted or unsubstituted heteroalkyl, R 8B -substituted or unsubstituted cycloalkyl, R 8B -substituted or unsubstituted heterocycloalkyl, R 8B -substituted or unsubstituted aryl, or R 8B -substituted or unsubstituted heteroaryl. 8B are independently halogen, -CN, -CF3, -CCl3, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, R 8C -substituted or unsubstituted alkyl, 8 4C -substituted or unsubstituted heteroalkyl, R 8C -substituted or unsubstituted cycloalkyl, R 8C -substituted or unsubstituted heterocycloalkyl, R 8C -substituted or unsubstituted aryl, or R 8C -substituted or unsubstituted heteroaryl. 8C is independently halogen, -CN, -CF3, -CCl3, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

[0209] In another embodiment, there is provided a compound of formula (II) as disclosed above, with the proviso that: (i) the compound of formula (II) is

[0210] [ka]

[0211] Instead, here, R 5is p-fluorophenyl or p-methylphenyl; (ii) the compound is

[0212] [ka]

[0213] Instead, here, R 6 is unsubstituted aryl, unsubstituted cyclohexyl, unsubstituted thiazole, or -CH-furanyl; or (iii) R 7 is not hydrogen.

[0214] In addition to any aspect disclosed above, in one embodiment, R 5 is not a substituted phenyl. 5 is not p-fluorophenyl and p-methylphenyl.

[0215] In one embodiment, the compound is R 6 In one embodiment, the compound does not have the structure of formula (IIa) where R 6 is p-fluorophenyl or p-methylphenyl.

[0216] In addition to any aspect disclosed above, in one embodiment, R 6 is not substituted or unsubstituted aryl, unsubstituted cyclohexyl, unsubstituted thiazole, and -CH-furanyl. 6 is an alkyl substituted with substituted or unsubstituted aryl, substituted or unsubstituted cyclohexyl, substituted or unsubstituted thiazole, or substituted or unsubstituted furanyl. 6 is not unsubstituted aryl, unsubstituted cyclohexyl, unsubstituted thiazole, and -CH2-furanyl.

[0217] In addition to any aspect disclosed above, in one embodiment, R 5is substituted or unsubstituted cycloalkyl or substituted or unsubstituted aryl. In one embodiment, R 5 is unsubstituted cycloalkyl or unsubstituted aryl.

[0218] In one embodiment, R 5 is substituted or unsubstituted C6-C8 cycloalkyl or substituted or unsubstituted phenyl. 5 is a substituted or unsubstituted C6, cycloalkyl, or substituted or unsubstituted phenyl.

[0219] In one embodiment, R 5 is R 5A -substituted or unsubstituted C6 cycloalkyl or R 5A substituted or unsubstituted phenyl, where R 5A is halogen. In one embodiment, R 5 is R 5A - substituted or unsubstituted phenyl, where R 5A is halogen. In one embodiment, R 5 is R 5A - substituted or unsubstituted phenyl, where R 5A is fluoro. In one embodiment, R 5 is unsubstituted phenyl.

[0220] In addition to any aspect disclosed above, in one embodiment the compound comprises R 6 is an alkyl substituted with substituted or unsubstituted aryl, substituted or unsubstituted cyclohexyl, substituted or unsubstituted thiazole, or substituted or unsubstituted furanyl.

[0221] In one embodiment, R 6 is a substituted or unsubstituted C4-C 12 Cycloalkyl, substituted or unsubstituted C3-C 12 In one embodiment, R is an alkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl. 6 is a substituted or unsubstituted C4-C12 Cycloalkyl, substituted or unsubstituted C4-C 12 In one embodiment, R is an alkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl. 6 is a substituted or unsubstituted C4-C 12 Cycloalkyl, substituted or unsubstituted C4-C 12 In one embodiment, R is a branched alkyl, or a substituted or unsubstituted phenyl. 6 is R 6A -Substituted or unsubstituted C4~C 12 Cycloalkyl, R 6A -Substituted or unsubstituted C4~C 12 Branched alkyl, or R 6A substituted or unsubstituted phenyl, where R 6A is halogen. In one embodiment, R 6 is R 6A -Substituted or unsubstituted C4~C 12 Cycloalkyl, R 6A -Substituted or unsubstituted C4~C 12 Branched alkyl, or R 6A -substituted or unsubstituted phenyl, where R 6A is fluoro. In one embodiment, R 6 is unsubstituted C4-C 12 Cycloalkyl, unsubstituted C4-C 12 Branched alkyl, or R 6A -substituted or unsubstituted phenyl, where R 6A is fluoro. In one embodiment, R 6 is unsubstituted C6-C 12 Cycloalkyl, unsubstituted C4-C 12 In one embodiment, R 6 is unsubstituted C6-C 10 In one embodiment, R 6 is unsubstituted C-C cycloalkyl. In one embodiment, R 6 is unsubstituted cyclohexyl.

[0222] In one embodiment, R7 is hydrogen or substituted or unsubstituted alkyl. In one embodiment, R 7 is hydrogen or unsubstituted alkyl. In one embodiment, R 7 is hydrogen or unsubstituted C1-C3 alkyl. 7 is hydrogen, methyl, or ethyl. 3 is methyl. In one embodiment, R 7 is ethyl. In one embodiment, R 7 is hydrogen.

[0223] In one embodiment, zl is 0, 1, 2, 3, or 4. In one embodiment, zl is 0 or 1. In one embodiment, zl is 0. In one embodiment, zl is 1. In one embodiment, z2 is 0, 1, 2, 3, 4, or 5. In one embodiment, z2 is 1.

[0224] In one embodiment, R 8 is independently substituted or unsubstituted alkyl. In one embodiment, R 8 is independently substituted alkyl. In one embodiment, R 8 is independently unsubstituted alkyl. In one embodiment, R 8 is independently substituted or unsubstituted heteroalkyl. In one embodiment, R 8 is independently substituted heteroalkyl. In one embodiment, R 8 is independently unsubstituted heteroalkyl. In one embodiment, R 8 is independently substituted or unsubstituted aryl. In one embodiment, R 8 is independently substituted or unsubstituted heteroaryl.

[0225] [ka]

[0226] For formula (IIc) (above), R 6is substituted or unsubstituted alkyl, or substituted or unsubstituted cycloalkyl; R 7 is substituted or unsubstituted alkyl. In one embodiment, R 6 is unsubstituted cycloalkyl (e.g., cyclohexyl, cycloheptyl, or cyclooctyl). 6 is unsubstituted alkyl (e.g., 3,3-dimethylbutyl). 7 is unsubstituted alkyl. In one embodiment, R 10 is an alkyl ester.

[0227] In another embodiment, the compound of formula (IId)

[0228] [ka]

[0229] Compounds having the formula:

[0230] For formula (IId), L 2 is a linker, B 1 is a purine base or an analogue thereof.

[0231] In one embodiment, L 2 is substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene. 2 includes water-soluble polymers. "Water-soluble polymer" means a polymer that is sufficiently soluble in water under physiological conditions (e.g., temperature, ionic concentration, etc.) to be useful in the methods described herein, as known in the art. An exemplary water-soluble polymer is polyethylene glycol.

[0232] In one embodiment, the water soluble polymer is -(0C2CH2)m-, where m is 1 to 100. 2A "cleavable element" is a chemical functional group that can undergo cleavage (e.g., hydrolysis) to release a compound, and the linker L 2 and optionally B1, and a linker L 2 Optionally, the residue 20 of

[0233] [Table 1]

[0234] [Table 2] JPEG0007638528000025.jpg209169JPEG0007638528000026.jpg206169JPEG0007638528000027.jpg139169

[0235] [Table 3]

[0236] Route and Formulation The administration of the composition with one or more antigens and one or more adjuvants, as well as the composition with any other active agent, or the administration of the composition with one or more antigens and one or more adjuvants, may be via any suitable route of administration (particularly parenteral, e.g., intravenous, intraarterial, intraperitoneal, intrathecal, intracerebroventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, or subcutaneous). Such administration may be a single bolus injection, multiple injections, or short-term or long-term infusion. Implantable devices (e.g., implantable infusion pumps) may also be used to parenterally deliver a particular formulation over time, at equal or varying doses. For such parenteral administration, the compound (conjugate or other active agent) may be formulated as a sterile solution in water or another suitable solvent or mixture of solvents. The solution may contain other substances, such as salts to make the solution isotonic with blood, sugars (particularly glucose or mannitol), buffers such as acetate, citrate, and / or phosphate, as well as their sodium salts, and preservatives.

[0237] The compositions of the present invention, alone or in combination with other active agents, can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to a selected route of administration, e.g., oral or parenteral (intravenous, intramuscular, topical, or subcutaneous routes).

[0238] Thus, the compositions can be administered systemically (e.g., orally in combination with a pharma- ceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier), alone or in combination with other active agents (e.g., antigens). They can be enclosed in hard or soft gelatin capsules, compressed into tablets, or added directly to the food of the patient's diet. For oral therapeutic administration, the compositions, optionally in combination with the active compounds, can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the active compound. The percentage of the compositions and preparations can, of course, be varied and can conveniently vary between about 2 and about 60% of the weight of a given unit dosage form. The amount of the conjugate and any other active compounds in such useful compositions is such that an effective dosage level will be obtained.

[0239] Tablets, troches, pills, capsules, etc. may also contain: binders such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, wintergreen oil, or cherry flavoring. When the unit dosage form is a capsule, it may contain a liquid carrier such as vegetable oil or polyethylene glycol in addition to the above materials. Various other ingredients may be present as coatings or may be present to modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, etc. Syrup or elixir may contain active compound, sucrose or fructose as sweetener, methyl and propyl paraben as preservative, coloring agent, and flavoring agent such as cherry or orange flavor.Of course, any ingredient used to prepare any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amount used.Furthermore, phospholipid conjugate, optionally in combination with another active compound, may be incorporated into sustained release preparations and devices.

[0240] The composition can also be administered intravenously or intraperitoneally by infusion or injection, optionally in combination with another active compound. Solutions of antigen(s) and adjuvant(s), optionally in combination with another active compound or its salt, can be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, and oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0241] Pharmaceutical dosage forms suitable for injection or infusion include sterile aqueous solutions or dispersions, or sterile powders containing the active ingredient (optionally encapsulated in liposomes) that are suitable for the extemporaneous preparation of sterile injectable or injectable solutions or dispersions. In all cases, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. Liquid carriers or vehicles can be solvents or liquid dispersion media, including, for example, water, ethanol, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The appropriate fluidity can be maintained, for example, by the formation of liposomes, the maintenance of the required particle size in the case of dispersions, or the use of surfactants. Prevention of the action of microorganisms during storage can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it can be beneficial to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0242] Sterile injectable solution is prepared by incorporating the required amount of compound in a suitable solvent with various other ingredients as listed above as necessary, followed by filtration sterilization.For the case of sterile powder for preparing sterile injectable solution, one method of preparation includes vacuum drying and freeze-drying technology, which produces a powder of active ingredient + any additional desired ingredients that exist in the solution previously sterile filtered.

[0243] For topical administration, the antigen(s) and adjuvant(s), optionally combined with another active compound, may be applied in pure form (e.g., they are liquids). However, it is generally desirable to administer them to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which may be solid or liquid.

[0244] Useful solid carriers include finely divided solids (e.g., talc), clay, microcrystalline cellulose, silica, alumina, etc. Useful liquid carriers include water, alcohol or glycol, or water-alcohol / glycol blends, in which the compounds of the present invention can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and antibacterial agents can be added to enhance the properties for a given use. The resulting liquid compositions can be applied by absorbent pads used in impregnated dressings and other bandages, or can be sprayed onto the diseased area using pump-type or aerosol sprayers.

[0245] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified minerals may also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for direct application to the user's skin.

[0246] Further, in one embodiment, the present invention provides various dosage formulations of antigen(s) and adjuvant(s), optionally in combination with another active compound, for inhalation delivery. For example, the formulations may be designed for aerosol use in devices such as nebulizers, dry powder inhalers, and nebulizers.

[0247] Examples of useful dermatological compositions that can be used to deliver compounds to the skin are known in the art, see, e.g., Jacquet et al. (US Pat. No. 4,608,392), Geria (US Pat. No. 4,992,478), Smith et al. (US Pat. No. 4,559,157) and Wortzman (US Pat. No. 4,820,508).

[0248] Useful dosage can be determined by comparing their in vitro activity and in vivo activity in animal models.Methods for extrapolating effective dosages in mice and other animals to humans are known in the art; see, for example, U.S. Patent No. 4,938,949.The ability of adjuvants to act as TLR agonists can be determined using pharmacological models well known in the art, including the procedure disclosed by Lee et al., Proc. Natl. Acad. Sci. USA, 100: 6646 (2003).

[0249] Generally, the concentration of phospholipid, optionally in combination with another active compound, in a liquid composition such as a lotion is about 0.1-25% by weight, e.g., about 0.5-10% by weight, and in a semi-solid or solid composition such as a gel or powder is about 0.1-5% by weight, e.g., about 0.5-2.5% by weight.

[0250] By administering the active ingredient, a peak plasma concentration of the active compound of about 0.5 to about 75 μM, for example, about 1 to 50 μM (such as about 2 to about 30 μM) can be achieved. This can be achieved, for example, by intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1 to 100 mg of the active ingredient. Desirable blood levels can be maintained by continuous infusion to provide about 0.01 to 5.0 mg / kg / hr, or by intermittent infusions containing about 0.4 to 15 mg / kg of the active ingredient.

[0251] The amount of antigen and adjuvant required for use in treatment, optionally in combination with another active compound, or active salt or derivative thereof, will vary depending on the particular salt selected, as well as the route of administration, the nature of the condition being treated, and the age and condition of the patient, and is ultimately at the discretion of the attending physician or clinician. In general, however, suitable dosages are in the range of about 0.5 to about 100 mg / kg, e.g., about 10 to about 75 mg / kg body weight / day (such as 3 to about 50 mg / kg of recipient body weight / day), e.g., 6 to 90 mg / kg / day, e.g., 15 to 60 mg / kg / day.

[0252] The antigen(s) and adjuvant(s), optionally in combination with another active compound, may conveniently be administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently 50 to 500 mg of active ingredient per unit dosage form.

[0253] The desired dose may conveniently be presented as a single dose or as divided doses administered at appropriate intervals (e.g., two, three, four or more times per day as sub-doses. The sub-dose itself may be further divided into a number of separate loosely spaced administrations (e.g., multiple inhalations from an inhaler or multiple applications of drops to the eye, etc.). The amount administered, and perhaps frequency of administration, will also vary according to the age, weight, condition, and response of the individual patient. In general, the active agent for the conditions described herein is The total daily dosage range of may be from about 50 mg to about 5000 mg, in single or divided doses. In one embodiment, the daily dosage should be in the range of about 100 mg to about 4000 mg (e.g., about 1000-3000 mg, in single or divided doses, e.g., 750 mg of the compound administered orally every 6 hours). This may achieve a plasma level of about 500-750 uM, which may be effective in killing cancer cells. In managing the patient, treatment should be initiated at a low dosage and increased depending on the patient's overall response.

[0254] Specific antigens may include amino acids, carbohydrates, peptides, proteins, nucleic acids, lipids, body substances, or cells such as microorganisms.

[0255] The specific peptide has from 2 to about 20 amino acid residues.

[0256] Another specific peptide has from 10 to about 20 amino acid residues.

[0257] Specific antigens include carbohydrates.

[0258] The specific antigen is a microorganism. The particular microorganism may be a virus, a bacterium, or a fungus.

[0259] Specific bacteria include Bacillus anthracis, Listeria monocytogenes, Francisella tularensis, Salmonella, or Staphylococcus. Specific Salmonella include S. typhimurium or S. enteritidis. Specific Staphylococcus include S. aureus.

[0260] Specific viruses are RNA viruses, including RSV and influenza viruses, products of RNA viruses, or DNA viruses, including herpes viruses. A specific DNA virus is the Hepatitis B virus.

[0261] The present invention includes compositions comprising phospholipid conjugates of TLR4 agonists and TLR7 agonists, optionally in combination with other active agents that may or may not be antigens (e.g., ribavirin, mizoribine, and mycophenolate mofetil).

[0262] Exemplary Embodiments In one embodiment, a method for enhancing an immune response in a mammal is provided.

[0263] In one embodiment, the method comprises administering to a mammal in need thereof a composition comprising an effective amount of a TLR4 agonist and a TLR7 agonist.

[0264] In one embodiment, the composition is a liposomal composition.

[0265] In one embodiment, the composition comprises a liposome comprising a TLR4 agonist and a liposome comprising a TLR7 agonist.

[0266] In one embodiment, the composition comprises a liposome comprising a TLR4 agonist and a TLR7 agonist.

[0267] In one embodiment, the TLR4 agonist and the TLR7 agonist are administered simultaneously.

[0268] In one embodiment, the TLR4 agonist has the formula (II):

[0269] In one embodiment, the TLR4 agonist comprises 1Z105, 2B182c, INI-2004, or CRX601.

[0270] In one embodiment, the TRL4 agonist is not 1Z105. In one embodiment, the TLR7 agonist has the formula (I).

[0271] In one embodiment, the liposome comprises PC, DOPC, or DSPC.

[0272] In one embodiment, the liposome comprises cholesterol.

[0273] In one embodiment, the method further comprises administering one or more immunogens.

[0274] In one embodiment, the immunogen is a microbial immunogen, such as one or more microbial proteins, glycoproteins, saccharides and / or lipopolysaccharides.

[0275] In one embodiment, the microorganism is a virus, such as influenza or chickenpox, or a bacteria.

[0276] In one embodiment, the microorganism is a parasite or a fungus.

[0277] In one embodiment, the liposome comprises one or more immunogens.

[0278] In one embodiment, the composition comprises one or more immunogens.

[0279] In one embodiment, the mammal is a human.

[0280] In one embodiment, the mammal is a rodent, equine, bovine, caprine, canine, feline, porcine or ovine.

[0281] In one embodiment, the amount of the TLR7 agonist is about 0.01 to 100 nmol, about 0.1 to 10 nmol, or about 100 nmol to about 1000 nmol.

[0282] In one embodiment, the amount of the TLR4 agonist is about 2 to 20 umol, about 20 nmol to 2 umol, or about 2 umol to about 100 umol.

[0283] In one embodiment, the ratio of TLR7 to TLR4 agonist is about 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200.

[0284] In one embodiment, the composition is injected.

[0285] In one embodiment, the liposome comprises DOPC and cholesterol.

[0286] In one embodiment, the immunogen is a cell, a protein, or a spore. In one embodiment, the immunogen is administered before or after the composition. In one embodiment, the administration is effective to prevent microbial infection. In one embodiment, the composition is administered intranasally. In one embodiment, the composition is administered intradermally.

[0287] In one embodiment, a pharmaceutical formulation is provided that includes a liposome, a TLR4 agonist, and a TLR7 agonist. In one embodiment, the liposome is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), 1,2-dioleoyl-3-trimethylammonium-propane (18:1DOTAP), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG ... 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:0 PEG-2000PE), 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino] lauroyl]-sn-glycero-3-phosphocholine (18:1 to 12:0 NBD PC), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-glycero-3-phosphocholine (16:0 to 12:0 NBD PC), and mixtures thereof; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, or mixtures thereof. In one embodiment, the liposome comprises DOPC, cholesterol, or a combination thereof. In one embodiment, the amount of the TLR7 agonist is about 0.01 to 100 nmol, about 0.1 to 10 nmol, or about 100 nmol to about 1000 nmol. In one embodiment, the amount of the TLR4 agonist is about 2 nmol to 20 umol, about 20 nmol to 2 umol, or about 2 umol to about 100 umol.In one embodiment, the ratio of TLR7 to TLR4 agonist is about 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200. In one embodiment, the TLR7 agonist comprises a compound of formula (I). In one embodiment, formula (I) is:

[0288] [ka]

[0289] where R 11 and R 12 are each independently hydrogen or an acyl group; R 13 is a negative charge or hydrogen, m is 1 to 8, the wavy line indicates the position of a bond, and OR 12 The absolute configuration at the carbon atom bearing is R, S, or any mixture thereof. In one embodiment, m is 1. In one embodiment, R 11 and R 12 are oleoyl groups.

[0290] In one embodiment, R 3 The phospholipids of R include two carboxylic acid esters, each of which includes one, two, three, or four sites of unsaturation, epoxidation, hydroxylation, or a combination thereof. 3 The phospholipids of X comprise two carboxylic acid esters, which may be similar or different. In one embodiment, each carboxylic acid ester of the phospholipid is a C17 carboxylic acid ester having an unsaturated site at C8-C9. In one embodiment, each carboxylic acid ester of the phospholipid is a C18 carboxylic acid ester having an unsaturated site at C9-C10. In one embodiment, X 2 is a bond or a chain having 1 to about 10 atoms in the chain, where the atoms in the chain are selected from the group consisting of carbon, nitrogen, sulfur, and oxygen, and any carbon atom may be substituted with oxo and any sulfur atom may be substituted with one or two oxo groups. 2 is C(O),

[0291] [ka]

[0292] It is.

[0293] In one embodiment, R 3 comprises dioleoylphosphatidylethanolamine (DOPE). In one embodiment, R 3 is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and X 2 is C(O). In one embodiment, X 1 is oxygen. In one embodiment, X 1 is sulfur, or -NR c where R c is hydrogen, C 1~6 Alkyl or substituted C 1~6 alkyl, where the alkyl substituents are hydroxy, C 3~6 Cycloalkyl, C 1~6 In one embodiment, X is alkoxy, amino, cyano, or aryl. 1 is -NH-. In one embodiment, R 1 and R c together form a heterocyclic ring or a substituted heterocyclic ring. 1 and R c together form a substituted or unsubstituted morpholino, piperidino, pyrrolidino, or piperazino ring. In one embodiment, R 1 is C1-C10 alkyl substituted with C1-6 alkoxy. 1 is hydrogen, C 1~4 Alkyl or substituted C 1~4 In one embodiment, R 1 is hydrogen, methyl, ethyl, propyl, butyl, hydroxyC 1~4 Alkylene, or C 1~4 Alkoxy C 1~4 In one embodiment, R 1is hydrogen, methyl, ethyl, methoxyethyl, or ethoxyethyl. 2 is halogen or C 1~4 alkyl or R 2 In one embodiment, R 2 is chloro, bromo, methyl, or ethyl, or R 2 In one embodiment, X 1 is O and R 1 is C 1~4 Alkoxy-ethyl, n is 0, and X 2 is carbonyl and R 3 is 1,2-dioleoylphosphatidylethanolamine (DOPE). In one embodiment, the compound of formula (I) is:

[0294] [ka]

[0295] It is.

[0296] In one embodiment, the compound of formula (I) is

[0297] [ka]

[0298] It is.

[0299] In one embodiment, in formula (II), z2 is 1, 2, or 3. In one embodiment, in formula (II), z1 is 1 or 2.

[0300] In one embodiment, in Formula (II), z1 is 0.

[0301] In one embodiment, in formula (II), R 5 is a substituted or unsubstituted aryl, or heteroaryl, e.g., unsubstituted C5 or C6 aryl.

[0302] In one embodiment, in formula (II), R 6 is a substituted or unsubstituted cycloalkyl, or heterocycloalkyl, e.g., 5, 6, or 7 cycloalkyl.

[0303] In one embodiment, in formula (II), R 7 is substituted or unsubstituted alkyl, for example, C1 to C5 alkyl.

[0304] In one embodiment, in formula (II), R 8 is a substituted or unsubstituted aryl, or heteroaryl, for example, a heteroaryl with 5, 6, or 7 atoms (such as furanyl, pyrrolyl, or imidazolyl).

[0305] The invention is further illustrated by the following non-limiting examples.

[0306] Example 1 Adjuvant potency of liposomally formulated 2B182c, a TLR4 agonist, and 1V270, a TLR7 agonist. Liposomal formulations of 2B182c (200 nmol / injection) and 1V270 (1 nmol / injection) alone or a combination of 200 nmol 2B182c and 1 nmol 1V270 were prepared (Inimmune Corp, Missoula, MT). The adjuvant potency of the liposomally formulated adjuvants was compared to a DMSO formulation (10% DMSO). The formulated adjuvants were tested using the same protocol. Briefly, female BALB / c mice were immunized with inactivated influenza virus on days 0 and 21 with liposomally formulated 2B182c (200 nmol / injection) and / or 1V270 (1 nmol / injection). Sera were evaluated for anti-HA and anti-NA antibodies (IgM, IgG1, and IgG2a) by ELISA. To determine whether formulated agonists affect germinal center B cell and plasmablast (antigen secreting cell) populations, inguinal lymph nodes were harvested and analyzed for B cell populations by FACS.

[0307] TLR4 is present on both the cell surface and in endosomal compartments. Signaling through endosomal receptors inhibits LPS-induced activation of NF-κB. Endosomal TLR4 activation induces TRIF pathway activation, leading to the release of type I IFN via IRF3 activation. Thus, the adjuvant activity of 2B182c may be attenuated by liposomal formulation. Liposomally formulated 2B182c induced significantly higher anti-HA IgG2a in mice immunized with 2B182c alone or 2B182c plus 1V270 compared with DMSO-formulated adjuvant, while liposomal 2B182c reduced HA- and NA-specific IgG1 (Figure 19A). Liposomal formulation did not affect IgG2a levels in 2B182c and the combined adjuvant of 2B182c and 1V270 (Figure 19A). The reduction in IgG1 levels by liposomal formulations is due to the Th1-biased immune response by liposomally formulated 2B182c and the 2B182 / 1V270 composite adjuvant (Figure 19B). These data are consistent with reports that intracellular delivery of TLR4 ligands induces effective Th1 immune responses in a type I IFN-dependent manner.

[0308] After antigen exposure, activated naive and memory B cells proliferate and mature in germinal centers (GCs). Maintenance of high antigen-specific Ab titers required for long-term vaccine efficacy correlates with GC formation. Activated B cells further form antigen-specific Ab secreting cells (ASCs; plasmablasts and plasma cells), memory B cells and other subsets. Plasmablasts were induced after seasonal influenza virus vaccination and peaked sharply on day 7 after vaccination. The frequency of plasmablasts in peripheral blood after vaccination with inactivated virus correlates with the degree of protective hemagglutinin inhibition title in humans. Therefore, GC B cells and plasmablasts in draining lymph nodes were examined. The number of germinal center B cells and plasmablasts was increased by the combination of liposomal 2B182c and 1V270 (Figure 20).

[0309] In summary, liposomally formulated TLR4 and TLR7 ligand adjuvants induced Th1 biased immune responses and increased GC-centered B cells and plasmablasts. To evaluate the quality of B cell responses induced by the combined adjuvants in liposomal formulations, we are currently performing BCR and TCR repertoire analysis of lymph node cells. Furthermore, functional evaluation of vaccine adjuvants will be evaluated by live virus (homologous and heterologous challenge).

[0310] Example 2 A combination of synthetic small molecule TLR4 and TLR7 agonists is a potent adjuvant of recombinant influenza virus hemagglutinin and induces rapid and sustained immunity that is protective against influenza virus in homogeneous, heterogeneous, and heterosubtypic mouse challenge models. However, the TLR4 agonist used in these studies was a first-generation leading synthetic TLR4 agonist of the pyrimidoindole class, 1Z105, optimized from hits identified in a high-throughput screening campaign to discover adjuvants that act as innate immune receptor agonists. 1Z105 was found to have good immune activity in mouse cells, but no significant activity in human cells. More recent studies have shown that a second-generation series of compounds containing C8-aryl substituents were more potent than 1Z105 in mouse cells, but were also highly active in human cells. Within this active group of C8-aryl derivatives, the C8-furan-2-yl derivative (2B182C) was selected for further study based on potency and favorable preformulation data (Figures 34 and 36). For comparison, the pyrimidoindole 2B182C was evaluated in combination with 1V270. The MPLA analog (MPLA-1), a potent TLR4 agonist, showed good protection against homologous and heterologous influenza challenge in vivo.

[0311] The TLR7 agonist, 1V270, is a lipid conjugate of a known TLR7 agonist. The main advantages of the agonist conjugate over the corresponding unconjugated agonist conferred by the lipid moiety of the agonist conjugate include greater potency and the absence of local or systemic toxicity often observed as cytokine syndrome. These favorable properties demonstrating efficacy and safety support the selection of 1V270 as a major TLR7 agonist for the combination adjuvant studies described in this technical proposal.

[0312] As described above, a composite adjuvant containing the TLR4 agonist 1Z105 and the TLR7 agonist 1V270 induced a broad defensive response with an influenza virus vaccine. In SAR studies, 2B182C, which showed higher agonist potency than 1Z105, was obtained in THP-1 cells and primary human and mouse cells in vitro. In a vaccine model using inactivated influenza virus [A / California / 04 / 09 (Cal / 09)], the adjuvant potency of 2B182C was examined and compared with 1Z105. These studies were conducted using a simple DMSO-water formulation of the TLR agonist.

[0313] <Composite adjuvants using TLR4-agonist and TLR7-agonist induce a rapid and broad defensive immune response against influenza virus infection> To evaluate the profile of the defensive immune response against influenza virus infection induced by the TLR4 / TLR7 agonist composite adjuvant, mice were immunized with a low dose (0.2 μg / injection) of recombinant hemagglutinin (rHA), humoral response, and protection against a lethal virus challenge (Figures 34A-2D). Mice immunized with rHA using the composite adjuvant showed minimal weight loss and higher survival rates (Figures 34B and 2C). The composite adjuvant and 1V270, the TLR7 agonist alone, induced a Th1-biased immune response.

[0314] To further investigate whether the TLR4 / TLR7 conjugate adjuvant provides cross - protection against heterologous influenza virus challenge, mice were immunized with 2009 - 2010 Fluzone containing B / Brisbane / 60 / 2008 (Victoria lineage) and challenged with 25 mL of D50 of the heterologous mouse - adapted virus B / Florida / 04 / 2006 (Yamagata lineage). More than 90% of the mice survived after vaccination with Fluzone adjuvant using 1V270 alone or in combination with 1Z105 (Figure 34E - 2G). These data indicate that 1V270, alone or in combination with 1Z105, induces a rapid and cross - protective immunity against heterologous influenza viruses.

[0315] <Determination of doses for TLR4 - agonist and TLR7 - agonist> As described above, in the SAR study, 2B182C, which showed higher potency in vitro compared to 1Z105 in human and mouse immune cells, was obtained. To investigate whether the higher potency observed in vitro is reproducible in vivo, female Balb / c mice were immunized intramuscularly (IM) on days 0 and 21 with inactivated influenza virus (A / California / 04 / 2009 (H1N1)pdm09, Cat# NR - 49450, BEI Resources) using TLR4 agonists (1Z105 or 2B182C, 40 or 200 nmol / injection) and 1V270 (lipid - TLR7 agonist conjugate, 0.2 or 1 nmol / injection) (Figure 34A). Serum was collected on day 28, and anti - hemagglutinin (HA) and anti - neuraminidase (NA) antibodies (IgM, IgG1, and IgG2a) were measured by ELISA. 1V270, 1Z105, and 2B182C were dissolved in DMSO, diluted, and the final concentration of DMSO was used at 10% as a vehicle control. Data were pooled from four independent experiments showing similar results.

[0316] <Effect of TLR agonist monotherapy on antibody secretion> As single adjuvants, 0.2 nmol and 1 nmol 1V270, and 40 nmol and 200 nmol 2B182C or 1Z105 were compared (Figure 34B). Both TLR4 agonists, 1Z105 and 2B182C, induced significantly higher levels of IgG1 against HA and NA. For IgG2a induction, both 0.2 and 1 nmol / injection 1V270 significantly increased anti-HA (p<0.05). Meanwhile, at 200 nmol / injection, only 2B182C, but not 1Z105, enhanced anti-NA Abs (p<0.01) (Figure 34B). 2B182C and 1Z105 induced similar levels of HA-specific IgG2a. No differences in IgM responses were observed with either adjuvant treatment. These data support reports that TLR4 agonists increase IgG1 production, TLR7 agonists have effects on IgG2a secretion, and 2B182C exerts potency equivalent to or slightly greater than that of 1Z105 in vivo.

[0317] <Effect of combined treatment with 2B182C and 1V270 on antibody secretion> Next, the potency of the combined adjuvants was evaluated using DMSO-water formulations. Both combined adjuvants of 1V270 with 1Z105 and 2B182C improved the induction of IgG1 against both HA and NA. 2B182C enhanced IgG1 significantly higher than 1Z105 at both 40 and 200 nmol (p<0.05, Figures 35A and 35B). For IgG2a induction, 2B182C increased the levels of anti-HA and anti-NA Abs, whereas 1Z105 failed in most cases (Figures 35C and 35D). These adjuvants showed minimal effects on IgM release (Figures 35E and 35F).

[0318] To compare the antibody titers of all tested combinations, the average titers of IgG1 and IgG2a are plotted in Figure 36A. 200 nmol of 2B182C + 0.2 or 1 nmol of 1V270 showed the highest induction of both IgG1 and IgG2a (Figure 36A). Furthermore, to evaluate the Th1 / Th2 immune balance, the IgG2a:IgG1 ratio was calculated for individual animals (Figure 36B). 1 nmol of 1V270 significantly shifted the Th2-biased immune response induced by 1Z105 or 2B182C, indicating that 1V270 shifted the immune response towards Th1 (Figure 36B). In summary, from the above results, the combination of 200 nmol / injection of 2B182C and 1 nmol / injection of 1V270 induced the highest amounts of IgG1 and IgG2a, and a Th1-biased immune response was shown to be desirable for heterologous protection against influenza virus infection. Therefore, the inventors selected this combination for further preclinical formulation.

[0319] <Preliminary data with TLR4 agonist> In vivo evaluations are performed for MPLA-2, a sulfate analog of MPLA, combinations, and all major TLR agonists in nanoparticle formulations. During the NIAID adjuvant discovery and development contract, a potent TLR4 agonist was discovered. There, an additive was demonstrated, even though there was no synergistic enhancement of influenza-related cytokine production in vitro (hPBMC), and no enhancement of in vivo IgG2A antibody and HI titers with 1V270 in mice and pigs. The main weakness of MPLA-1 as an adjuvant is its lack of chemical stability as it is prone to hydrolysis in aqueous media. In preliminary mouse studies of non-specific resistance, MPLA-2 protected mice from a lethal influenza challenge better than an equal amount of MPLA-1. Therefore, MPLA-2 represents the next generation TLR4 agonist.

[0320] To support the objectives outlined above, the experiments detailed below are conducted.

[0321] <Research Area 1: Formulation and Analytical Assay Development of Combinations of Major TLR Agonists> <Development of Formulations of TLR4 / TLR7 Combinations> <Task 1A: Development of Colloidally Stable Nanoparticle Formulations of Major Compounds Alone and in Combinations> Particle delivery systems act as adjuvants by mimicking the size and shape of viral and bacterial pathogens that our immune system has evolved to recognize and fight through pattern recognition receptors (PRRs). Research over the past 30 years has led to a number of nanoparticle- and microparticle-based systems that are biodegradable and suitable for vaccine antigen delivery. Their usefulness as vaccine delivery systems has been demonstrated in the literature using liposomes, virosomes, Iscoms, emulsions, virus-like particles (VLPs), solid lipid nanoparticles (SLNs), and poly(lactic-co-glycolic acid) (PLGA) polymers, and examples of each type are in human clinical trials. The major adjuvant mechanism of particulate delivery vehicles is thought to be the enhanced uptake of particles or associated antigens taken up by APCs. The addition of PAMPs to antigens has now been well established to promote robust innate and adaptive immune responses through ligation of TLRs and other PRRs that lead to activation of innate immune cells. Many PAMPs (such as bacterial lipoproteins, glycolipids, DNA, and viral RNA) have been identified and isolated from viral and bacterial pathogens. Many of these agonists are potent adjuvants but result in unacceptable levels of inflammation or have unfavorable physical / chemical properties for clinical development. In response, researchers have successfully produced synthetic analogs with improved safety and chemical profiles, and many of these have been added to particle delivery systems to enhance their pathogen mimicry through PRR ligation. Particle delivery systems can also be used to improve the in vivo biodistribution kinetics of adjuvants and reduce the side effects of adjuvants without sacrificing the immunogenicity of the adjuvant.

[0322] The use of PEGylated liposomes with the TLR4 agonist MPLA-1 incorporated into the bilayer for effective sublingual vaccination has been demonstrated in a mouse model of influenza. This formulation reduces the pyrogenicity of MPLA-1 by 200-fold without any loss of adjuvant potency in vivo. This is similar to the reduction in pyrogenicity of LPS observed when incorporated into liposomes versus aqueous dispersions. Similar reductions in pyrogenicity are expected for TLR4 agonists.

[0323] A number of different lipids and components were evaluated for nanoparticle / microparticle formation, API uptake, API stability and colloidal stability. A series of commercially available cationic (DDA, DOTAP, DC cholesterol), anionic (DPG, PS, POPG) and neutral lipids (PC, DOPC, DSPC) are tested with TLR4 and TLR7 agonists. Other formulations may use PLGA, polycaprolactone, poly(propargyl methacrylate) or PLMA. Particle size and charge have been shown to significantly affect nanoparticle uptake and processing by DCs, so the impact of these variables to drive delivery vehicle design on the quality attributes listed above will be explored. Small scale liposome formulations can be prepared using thin film methods adapted to sterile serum vials to further reduce scale and waste.

[0324] Briefly, this is done by: 1. Add API to lipids and dissolve them in chloroform (fluorescent markers may be added at this step if desired, e.g. for NBD, BODIPY, FITC, etc.) 2. Rotary evaporation at a set speed and vacuum application to obtain a dry thin film 3. Rehydration with aqueous buffer (0.1 M phosphate, Tris or HEPES) 4. Particle size reduction by bath sonication above the lipid transition temperature (Tm) with in-process monitoring of particle size, polydispersity and surface charge (zeta potential) by dynamic light scattering (DLS) 5. Prepare the main formulation lots at 3-10 mL scale using a Lipex extruder, which improves particle size uniformity (polydispersity index, PDI) over the sonication method.

[0325] Task 1B: Stability testing to evaluate the colloidal and physical stability of formulations Formulation stability is necessary for successful commercial product development as it affects product storage, shipping, and shelf life, all of which directly contribute to product cost. Formulations are demonstrated to be suitable as potential products as well as stable, especially when selecting lead candidates for further pursuit.

[0326] Lead formulations are evaluated for short-term accelerated (25 and 40 °C) and long-term real-time stability (2-8 °C and 25 °C) to ensure that the selected formulation provides sufficient stability for potential product development (minimum of 12 months at preferred storage conditions).

[0327] Accurate quantification of adjuvant incorporation into nanoparticle delivery systems is essential for proper dosing, vaccine efficacy and safety. We developed SEC-HPLC and RP-HPLC methods for quantification of TLR4 and TLR7 / 8 agonists incorporated into nanoparticles, including liposomes. RP-HPLC is effective for analysis of total agonist content present in nanoparticles when samples are dissolved in water-miscible organic solvents (methanol, tetrahydrofuran, etc.) with sufficiently low background UV absorbance. Dissolution with organic solvents disrupts the nanoparticles, releasing the incorporated or surface-bound agonist for accurate quantification by RP-HPLC against a five-point standard curve.

[0328] Quantification of liposomally incorporated (bilayer or aqueous core) agonists requires a method capable of analyzing intact lipid liposomes as well as the aqueous phase outside the liposomes. We used a SEC-HPLC method capable of quantifying "free" TLR agonists with UV detection at 296, 225, and 310 nm (for 2B182C, MPLA-2, and 1V270, respectively). TSK gel SWx1 series columns provide excellent size-based resolution for nanoparticle formulations in the 30-200 nm range. The mobile phase used is the same as the buffer utilized for liposome rehydration to maintain a constant osmotic potential between the fluid outside the liposomes and the aqueous phase in the liposome core. This method was qualified as a complementary method to the in vitro potency assay, detecting only aqueous unincorporated TLR4 agonists.

[0329] A preliminary study was conducted using these analytical methods to evaluate liposomal formulations of 2B182C and 1V270, prepared alone and in combination (coencapsulated). The workflow was carried out as follows: 1) Lead adjuvant formulation screening at 2mL scale with target concentrations of 200nmol for 2B182C and 1nmol of 1V270 in 50uL for IM injection (pharmaceutical acceptable co-solvents, excipients, liposomes). 2) Perform basic analytical method development and analysis for major formulations to ensure that the formulations meet quality standards. 3) Evaluate the stability of the preferred formulations by real-time and accelerated methods and add appropriate excipients as stabilizers if necessary. 4) rFC testing to ensure the final product is free of endotoxin contamination.

[0330] All liposomal formulations were prepared at 2 mL scale for compounds 2B182C and 1V270.

[0331] Briefly, liposomes were prepared using the following procedure, and the following compositions were evaluated: 2B182 with and without 1V270 (2:1, with and without DOPC / cholesterol, respectively). The concentration of DOPC tested was kept constant at 40 mg / mL, resulting in a cholesterol concentration of 10 mg / mL. Liposomes were prepared according to the lipid film rehydration method using 9:1 chloroform:methanol as the solvent. The rehydration buffer used initially was 50 mM NaPB, 100 mM NaCl, pH=6.1. The agonist concentrations tested were the target concentrations. Sonication at elevated temperature was used to reduce the liposome particle size. A summary of the analytical results is shown in Table 4.

[0332] [Table 4]

[0333] [Table 5]

[0334] Evaluate other ratios of TLR agonist, other lipid components, and varying amounts of cholesterol for nanoparticle formation. At least 10 different formulations will be prepared and screened for suitability in the process under Task 2A and compared with the results obtained using the simple DMSO-water formulation described above.

[0335] Nanoparticles: TLR7 and TLR4 agonists are prepared as nanoparticle formulations (liposomes, SLN, PLGA, emulsions, etc.). The final lead formulation is selected based on immunology, stability and manufacturing data. DOPC / cholesterol liposomal formulation appears very promising based on preliminary immunology and stability data. One of the challenges anticipated with TLR4 and TLR7 agonists is the co-incorporation of both agonists into the same nanoparticle in a controlled and consistent manner. The ratio of agonists to each other is fixed once co-encapsulated and any dose adjustment at that point will alter both agonists together.

[0336] Analytical methods: All of the analytical methods described in Task 1C were used with our lipidated TLR-7 / 8 and TLR4 agonists, and we expect to further improve their specificity, linearity and range with further optimization. RP-HPLC methods for quantification of adjuvants in TLR4 and TLR7 agonist formulations are optimized for peak shape, LOD and LOQ. These same methods are gradient and column optimized to achieve baseline resolution and optimal LOD / LOQ for any degradants detected from stability studies to allow accurate monitoring of product stability. Accurate quantification of nanoparticle incorporation percentages for each agonist in a TLR4 and TLR7 agonist combination may prove difficult, as SECHPLC separates based solely on hydrodynamic volume. Liposomes and unincorporated agonists may have similar particle sizes, limiting the usefulness of SEC-HPLC for incorporation determination. This is discussed below in an alternative approach.

[0337] Alternative approach: If developing co-encapsulated TLR4 and TLR7 agonists proves too difficult due to inconsistent levels of agonist in the nanoparticles, our immunological data showed that adjuvant synergy could still be achieved by simply mixing a TLR7 agonist in liposomes with a TLR4 agonist in liposomes. This approach has the potential to generate a simpler and more reliable product whose analytical characterization would be easier by reducing the chance that the agonist signals would interfere with each other.

[0338] Another option would be to explore other formulations for co-encapsulation such as nanoemulsions where 100% of the agonist is incorporated by default as the aqueous and oil phases are mixed into the nanodroplets. Emulsions also have the advantage of forming a depot at the site of administration, which may further enhance the immune response. As discussed in research area 2, co-encapsulated TLR4 and TLR7 agonists and mixed agonists will be compared in vitro and in vivo to evaluate the advantages and disadvantages of these approaches. An alternative approach to using SEC-HPLC to determine agonist incorporation into nanoparticles would be high-speed density gradient centrifugation to pellet the nanoparticles and analyze the supernatant for unincorporated agonist using established RP-HPLC methods.

[0339] Formulations of the target ratio range with acceptable properties for advancement will be subjected to in vivo studies, including immunization and viral challenge studies.

[0340] <Research Area 2: Establishing immunological biomarkers of protection from lethal influenza virus challenge by major adjuvant formulations> Uncovering reliable biomarkers is necessary for the successful development of safe and effective vaccines. It is essential for vaccine development programs to select vaccine candidates with a profile that effectively prevents infection without any safety issues. Vaccine clinical trials require the identification of biomarkers that predict antigen-specific adaptive immune responses with minimal reactogenicity. In this project, biomarkers will be identified in two steps: 1) innate immune biomarkers induced by the primary adjuvant in the formulation with or without antigen, and 2) biomarkers that correlate with adaptive immune responses. Therefore, in vitro and in vivo studies will be performed to identify biomarker candidates that correlate with the biological activity of both TLR4 and TLR7 / 8 ligands and are also associated with reactogenicity.

[0341] <Task 2A: Combination formulations based on in vivo antibody production studies of immune activity and reactogenicity> A hallmark of vaccination-mediated protection from infectious diseases is the induction of effective antibody production. The combined use of TLR4 and TLR7 agonists significantly increased antigen-specific antibody titers. A trend toward a Th1 bias of the immune response was observed. The efficacy of formulated adjuvants and their combinations is compared to simple DMSO-water preparations.

[0342] Task 2A.1: Immunization studies in mice with key combo formulations The formulation of the primary adjuvant will be evaluated in an immunization study, alone and in combination with various ratios of TLR agonists, in a similar manner as previously completed for the DMSO-water formulation. Levels of IgM, total IgG, and IgG1 and IgG2a specific for both HA and NA will be evaluated. The ratio of one or more of the TLR agonists in the combination that provides the greatest titers of antigen-specific antibodies will be identified. This formulation will be carried forward to challenge studies under Research Area 3.

[0343] Task 2A.2: Reactogenicity and toxicity assessment of key combo formulations in mice Infectious disease vaccines are designed to be protective in healthy populations, so vaccine safety must be the highest priority among development goals. Therefore, appropriate experiments are performed to evaluate the toxicity and reactogenicity of the candidate formulation. In these experiments and in general, overt toxicity is rigorously evaluated as an initial toxicity assessment. Any signs of distress in the mice (i.e., lack of grooming, mobility problems, conjunctiva, abnormal behavior, reactivity, etc.) are noted. In addition to gross observations, toxicity measurements include complete blood counts, serum chemistry evaluations (AST, ALT, ALP, amylase, blood urea nitrogen, creatinine, total protein, glucose, potassium, calcium, sodium, total bilirubin) and necropsy evaluations (hematoxylin and eosin stained sections of spleen, liver and kidney). In addition, the injection sites are evaluated for visible signs of inflammation and other abnormal findings. As part of the necropsy evaluation, tissues at the injection site are also evaluated histologically. These studies are summarized in Table 6 below.

[0344] <Task 2B: Identification of immune markers that can predict protective adaptive immune responses> As previously mentioned, the identification of biomarkers that predict antigen-specific adaptive immune responses with minimal reactogenicity will facilitate clinical trial design and methods.

[0345] <Task 2B.1: Innate immune response signatures (cytokines, chemokines)> Immune cell recruitment by chemokines to the local vaccine administration site is essential to recruit antigen-presenting cells (APCs) and influence the subsequent induction of adaptive immune responses. However, the injection site, i.e., muscle tissue, contains a relatively small number of immune cells, so an effective adjuvant must induce the recruitment of immune cells to the local site. Unlike TLR7 / 8, TLR4 is abundantly expressed on non-immune cells and can express sufficient chemokines to recruit inflammatory cells. After TLR stimulation, the recruitment of APCs is usually accompanied by inflammatory cells, making it difficult to distinguish the inflammatory response from the adjuvant effect. These complex cascades of immune activation cannot be studied by in vitro assays alone. Therefore, a panel of markers is selected from the above in vitro experiments in samples obtained from in vivo studies in mice.

[0346] Primary adjuvant formulations are administered intramuscularly (IM) to mice and serum is collected on days 1, 3 and 7 post-injection to examine systemic cytokine / chemokine levels. Cytokine / chemokine and costimulatory molecule gene expression is examined by qPCR or NanoString assay as described in Task 2A.2 above for local muscle tissue. Immune cell infiltration is assessed by histological examination of selected samples with hematoxylin-eosin and immunohistochemical staining. Spleen cells or PBMCs are used to evaluate the expression of costimulatory molecules assessed by flow cytometry. Draining lymph nodes are harvested at the indicated time points, pooled for each experimental group and analyzed for immune cell populations and expression of chemokine receptors, costimulatory molecules. A summary of the study design is provided in Table 6. Note that the "Group 5: Combined Adjuvant with Antigen" group may contain combinations of TLR4 and TLR7 agonists in different ratios as needed to provide the desired profile of cytokine / chemokine induction. Innate immune signatures that exhibit biological activity of both TLR4 and TLR7 ligands and are also associated with reactogenicity are selected.

[0347] [Table 6]

[0348] <Task 2B.2: Adaptive immune response signatures> Experiments to evaluate adaptive immune responses will be performed in conjunction with Task 2A.1 above. Candidate biomarkers that meet the following criteria will be identified: 1) detectable in peripheral blood, 2) driven by the mechanism of action of each TLR ligand and correlate their biological effects, 3) predict long-term antigen-specific antibody induction and broad protection, and 4) predict reactogenicity.

[0349] Results and alternative approaches The ratio of one or more of the combined TLR agonists provides the maximum titer of antigen-specific antibodies. Moreover, the use of a combination of two classes of TLR ligands results in a shift of the adaptive immune response towards a Th1-biased response compared to the use of a TLR4 agonist alone. Thus, the Th1 / Th2 response ratio is likely to be increased. This Th1 bias may be advantageous to broaden the response to include heterologous virus protection. With regard to toxicity, systemic and oral administration of the imidazoquinoline class of TLR7 / 8 ligands has shown severe side effects including flu-like symptoms, nausea and lymphopenia accompanied by high levels of serum TNFα and IL-1β. This also applies to the oxoadenine class of which 1V270 is a member. However, most of these undesirable side effects can be avoided by using the usual local administration route for vaccination, IM. Moreover, TLR7 / 8 ligands have been prepared by conjugation to lipid moieties as well as by customizing formulations, successfully reducing systemic cytokine release while maintaining adjuvant activity. Therefore, there will be little to no reactogenicity associated with the combination of key formulations due to low systemic exposure to inflammatory cytokines.

[0350] <Research Area 3: Formulation Selection and Immunization - Virus Challenge Study in Mice (Inimmune)> For preclinical immunization / viral challenge studies in mice, a lead formulation combination will be selected along with a backup combination based on the results of the formulation studies, including stability (Task 1B), immune activity (Task 2A.1) and reactogenicity profile (Task 2A.2). Viral antigens used in the studies may be selected from either recombinant vaccine antigens or inactivated whole viruses used in licensed commercial vaccines such as A / Victoria / 3 / 75(H3N2), A / Michigan / 45 / 2015(H1N1)pdm09-like viruses and A / Hong Kong / 4801 / 2014(H3N2)-like viruses.

[0351] <Task 3A: Selection of major combo products> The primary selection criteria were based on 1) stability of the formulated combination, 2) the ratio of TLR agonists that provides the desired antigen-specific antibody levels, and 3) a low reactogenicity profile, both locally and systemically. Specific studies related to these criteria are summarized in Table 7.

[0352] Following selection of the lead formulated combination and the backup lead combination, evaluation of the selections in an immunization / viral challenge model in mice is performed (task 3B).

[0353] [Table 7]

[0354] <Task 3B: Primary formulation immunization / virus challenge studies> <Task 3B.1: Determining the Minimum Protective Dose for Virus Challenge Studies> Since inactivated influenza viruses contain innate immune receptor ligands (PAMPS), a certain low level of protection can be expected after immunization of mice with sufficient antigen alone. Therefore, studies are performed to determine the minimum protective dose (if any) using inactivated viruses. The minimum protective dose of antigen is the dose that provides only partial protection (less than 30% survival) upon subsequent challenge with a matched strain of active virus. This strategy allows a range of activity to be observed with the selected major formulated adjuvant combination. Furthermore, a dose of challenge virus can be identified that also results in complete mortality for unimmunized mice (typically a dose of about 5 LD50).

[0355] <Task 3B.2: Homologous virus protection research> Following antigen dose range-finding studies, a mouse model will be used to evaluate the immunogenicity of key adjuvant combinations with homologous influenza vaccine antigens. Key determinants of success will be: 1) persistent influenza-specific IgG2a and IgG1 in serum; 2) protection from lethal influenza virus challenge; 3) low reactogenicity; and 4) polyfunctional CD4+ as assessed by intracellular IFNγ / TNFα staining. + vs. CD8 + Secondary endpoints are the induction of T cells. Secondary endpoints are the scoring of disease severity by monitoring weight gain / loss and observable clinical symptoms (wavy coat, hunched posture and labored breathing) after vaccination or influenza virus challenge.

[0356] <General in vivo methods> Immunological evaluation: Mice (male and female) are vaccinated (adjuvant + influenza antigen such as A / Victoria / 3 / 75 (H3N2)) IM once or twice with 21 days between the first and second vaccination (Figure 12). Cellular immunity (CMI) is assessed by Th1 / Th2 cytokine induction in spleen cell cultures (analyzed by ELISA) and polyfunctional CD4 + and CD8 +T cell responses (analyzed by FACS, 10-color intracellular cytokine staining) were assessed in a subset of 4 mice per group. In addition, tetramer staining and cell surface phenotyping were performed to identify influenza-specific memory CD4 + and CD8 + T cell frequencies are measured. Influenza-specific humoral responses are measured in serum (IgG1 and IgG2a) and HI titers are used to measure functional antibody titers. Groups of vaccinated and control mice were challenged with 5 LD50 of A / HK / 68(H3N2) and assessed for survival, weight gain, and disease severity over 21 days. Reactogenicity in these mouse studies is measured by weight loss and symptom scores and assessment of injection site infiltration. Differences in p-values ​​<0.05 are considered significant. Analysis of variance (ANOVA) and Tukey ANOVA are performed on all data to demonstrate robust statistical significance.

[0357] <Task 3B.3: Cross-virus protection research> Following the homologous protection studies, the same study design is used to evaluate the combination of key adjuvants in a mouse model of heterologous or heterosubtypic protection. Mice are immunized as above (Task 3B.2) but challenged with an influenza virus strain of a different HA / NA type (e.g. A / Puerto Rico / 8 / 1934 (H1N1)). Protection observed in such a challenge model suggests a broadening of the antigen-specific response to include antigens common to both strains, such as the stalk region of the HA protein. To confirm such a broadening, studies of the B cell receptor (BCR) and T cell receptor (TCR) sequences are performed.

[0358] Results and alternative approaches As mentioned above, a growing number of literature reports cite combinations of various TLR agonists that can synergistically increase the magnitude of vaccine-mediated immunity and alter the type of downstream adaptive immune response generated, thereby enhancing the efficacy of these vaccines. Adjuvant combinations for influenza virus challenge protection are described herein.

[0359] Example 3 <Influenza hemagglutinin (HA) as a vaccine antigen> Strategies to boost broadly neutralizing stalk antibodies include 1) focusing on headless HAs, which remove the entire head domain to make the stalk domain more “available” and thus induce antibody responses against the stalk domain, or 2) using a combination of chimeric HAs consisting of stalk domains from H1, H3 or influenza B viruses.

[0360] Immunization with one antigen is known to block a strong immune response to a second similar antigen ("antigenic original sin"). This is important for infectious diseases with repeated infections (influenza) or where there is antigenic evolution (HIV, malaria). For influenza, the primary neutralizing antibodies are made against the head region of the hemagglutinin (HA) of the virus. Different virus strains have different HA head regions, which cross-react weakly with antibodies but inhibit responses to new epitopes. For HIV, mutated epitopes on the virus do not stimulate antibodies or T cells due to epitope suppression.

[0361] The mechanism of antigenic original sin in vaccines could be due to epitope exclusion (pre-existing antibodies, especially mucosal IgA, shield the vaccine from all antigen-presenting cells (APCs)); dendritic cell access (memory B cells internalize the new vaccine, resulting in reduced DC activation and T cell immunity); and / or T cell competition (memory B cells become activated, consume cytokines, cofactors, and capture T cells that can react with antigen-loaded DCs).

[0362] To overcome antigenic sin in vaccines, dosage may be increased (e.g., larger vaccine doses (three times the amount of influenza vaccine given to patients over 60 years old)); encapsulation (placing the vaccine in an emulsion or liposome that delivers the vaccine preferentially to dendritic cells (Shingrix, chickenpox vaccine against shingles)); and / or dendritic cell activators (TLR agonists may increase the diversity of numbers of activated T cells against vaccine antigens).

[0363] To study antigenic original sin in mouse models, one can use: hapten-protein conjugates (haptens are small molecules such as fluorescein or DNP that can be conjugated to protein antigens such as ovalbumin and KLS); or pre-immunization with unconjugated protein antigens inhibits the antibody response to immunization with hapten-protein conjugates. For influenza in these models, one can hyperimmunize with one protein, such as influenza HA, for one virus strain and boost with a partially cross-reactive HA from another strain, then analyze the B and T cell immune responses to the second HA, including epitopes recognized, clonal diversity by nexgenRNA sequencing, and neutralization capacity, and subsequently correlate with in vivo protection.

[0364] Shingrix is ​​a recombinant VSV glycoprotein E, Salmonella-derived nonophosphoryl lipid A, and QS-21 saponin molecule in a liposomal formulation made from dioleoylphosphatidylcholine and cholesterol in buffered saline that is reconstituted at the time of use. To make the influenza vaccine similar to Shingrix, this vaccine has a protein antigen, two adjuvants in a liposomal formulation.

[0365] Example 4 The efficacy of annual influenza vaccines is still estimated at 10-60% due to antigenic drift of influenza viruses. Synthetic TLR4 and TLR7 agonists (1Z105 and 1V270) enhanced Th2- and Th1-mediated anti-hemagglutinin antibody production, respectively. The combination of 1Z105 with 1V270 promoted balanced Th1 / Th2 immunity to protect against influenza virus infection. To enhance the efficacy of the adjuvant, structure-activity relationship studies were performed on 1Z105 and 2B182C, which was the more potent derivative in vitro. In an in vivo vaccine study using the model antigen ovalbumin, 2B182C induced higher serum IgG1 levels and additionally enhanced the release of antigen-specific IgG2a induced by 1V270. Furthermore, liposomal formulations of 2B182C and 1V270 reduced cytotoxicity and reactogenicity and maintained activity in enhancing both Th1- and Th2-mediated antibody production. In an in vivo vaccination study with inactivated A / California / 04 / 2009(H1N1)(pdm09) as the antigen, the liposomal combination adjuvant increased the populations of T follicular helper cells, germinal center B cells, and antibody-secreting plasma cells. Next-generation sequencing analysis of B and T lymphocytes in draining inguinal lymph nodes showed that the combined adjuvant increased B cell clonotypes of immunoglobulin heavy chain (IGH) genes and shared B cell clonal and TCR clonality. These findings suggested that the combination contributed to enhancing antigen-specific Th1 immune responses. Finally, the vaccine with the combined adjuvant protected against a lethal homologous virus challenge with less lung damage.

[0366] <Method> <Mouse> Female 6- to 8-week-old BALB / c mice were purchased from Jackson laboratory (Bar Harbor, MA). Animal experiments were performed in the University of California, San Diego animal facility using ovalbumin or inactivated influenza virus as antigens and did not require live virus challenge. Influenza challenge studies were performed by the Animal Research Center at Utah State University using 6-week-old female BALB / c mice (Charles River Laboratories, Wilmington, MA). All animal experiments were approved in advance by the Institutional Animal Care and Use Committee (IACUC) of UC San Diego or Utah State University.

[0367] <Cells and Reagents> TLR4 / NF-kB reporter cell line HEK-Blue TM Human TLR4 and HEK-Blue TM Mouse TLR4 cells were purchased from InvivoGen (catalog no. San Diego, CA). Mouse primary BMDCs were prepared from bone marrow cells harvested from the femurs of C57BL / 6 mice. BMDCs were treated with the indicated compounds in RPMI supplemented with 10% FBS (Omega, Tarzana, CA) and penicillin / streptomycin (100 units / mL / 100 μg / mL, Thermo Fisher Scientific, Waltham, MA). Monophospholipid A (MPLA), AddaVax, was purchased from InvivoGen (catalog no. San Diego, CA). Inactivated influenza A virus [A / California / 04 / 2009(H1N1)pdm09] (IIAV) was obtained from BEI Resources (#NR-49450, Manassas, VA). The TLR7 agonist 1V270, the TLR4 agonist 1Z105 and its derivatives including 2B182C were synthesized. Liposome formulations of 1V270 (20 μM), 2B182C (4 mM) and 1V270 + 2B182C (20 μM + 4 mM) were made at Innimune corp. (Missoula, MT).

[0368] <TLR4 / NF-κB reporter cell assay> TLR4 / NF-κB activation was evaluated using HEK-Blue TM hTLR4 and HEK-Blue TM mTLR4 (InvivoGen). Cells were treated with 1Z105 and 2B182C (two-fold serial dilutions starting from 10 μM) for 20 h. NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) protein in the culture supernatant was measured according to the manufacturer's protocol.

[0369] <Evaluation of antibody production in vivo> BALB / c mice were immunized intramuscularly (i.m.) in the gastrocnemius muscle of the hindlimbs on days 0 and 21 with IAV (10 μg / injection) plus the indicated adjuvant. The detailed concentrations of the adjuvant and the number of animals in each treatment are described in the legends of each figure. Serum was collected on day 28 and evaluated for antigen-specific antibodies (anti-HA IgG1, anti-NA IgG1, anti-HA IgG2a, anti-NA IgG2a, anti-HA IgM, and anti-NA IgM). ELISA for these antibodies was performed as previously described (reference). For studies using DMSO formulations, 10% DMSO was used as the vehicle. In experiments using liposome-formulated adjuvants, 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol (DOPC / Chol, control liposomes) were used as the vehicle.

[0370] <NGS assay of BCR and TCR repertoires> The immunization protocol was shown in Figure 28A. Briefly, mice were sacrificed on day 28 and inguinal lymph nodes were harvested. Total RNA was extracted from lymphocytes (bulk) using RNeasy Mini Kit (Qiagen, Hilden, Germany), and RNA quality was confirmed by Agilent 4200 TapeStation (Agilent, Santa Clara, CA). Next-generation sequencing was performed using unbiased TCR repertoire analysis technology (Repertoire Genesis Inc., Osaka, Japan).

[0371] <Evaluation of protection against lethal influenza virus challenge> BALB / c mice were vaccinated im on day 0 with IIAV (3ug / injection) using formulations 1V270 and 2B182C and infected intranasally on day 21 with homologous or heterologous influenza A viruses, A / California / 04 / 2009 (pdmH1N1) and A / Victoria / 3 / 75 (H3N2), respectively. An immunizing dose of IIAV; 3ug / injection, that protected 30-50% of animals from homologous virus challenge was determined in preliminary experiments. For influenza virus challenge, groups of mice were anesthetized by intraperitoneal injection of ketamine / xylazine (50mg / kg / 5mg / kg) and then infected with 1x10 per mouse. 5 (3×LD 50 ) cell culture infectious dose (CCID 50 ) influenza A / California / 04 / 2009 (H1N1pdm) virus per mouse; 5 × 10 2 (3×LD 50 )CCID 50Mice were challenged intranasally with influenza A / Victoria / 3 / 75 (H3N2) virus in a 90 μL suspension. All mice were challenged with the virus on study day 21. Influenza virus (H1N1pdm) with strain designation 175190 was received from Dr. Elena Govorkova (Department of Infectious Diseases, St. Jude Children's jemResearch Hospital, Memphis TN). The virus was adapted to replication in the lungs of BALB / c mice by nine passages in mice. The virus was plaque purified in Madin-Darby canine kidney (MDCK) cells, and virus stocks were prepared by growth in embryonated chicken eggs followed by growth in MDCK cells. Influenza A / Victoria / 3 / 75 (H3N2) virus was obtained from the American Type Culture Collection (Manassas, VA). The virus was not initially lethal to mice, but became lethal after seven serial passages in the lungs of infected animals. After mouse adaptation, virus stocks were prepared by propagation in MDCK cells.

[0372] <Measurement of lung virus titers and lung inflammation> Six days after viral challenge, bronchoalveolar lavage (BAL) procedures were performed immediately after blood collection and were completed within 5–10 min of death for each animal. A volume of 0.75 mL of phosphate-buffered saline (PBS) was slowly delivered into the lungs through the tracheal tube. Immediately after delivery, the fluid was slowly withdrawn by gentle suction and samples were stored at -80 °C. This procedure was repeated a total of three times and lavage fluids from each mouse were pooled. To measure lung viral titers, BAL samples were centrifuged at 2000 g for 5 min. Various 10-fold dilutions of BAL supernatants were assayed in triplicate for infectious virus in MDCK cells and viral titers were calculated. For measurement of lung cytokine levels, samples (200 μL) from each lung lavage were tested for MCP-1 and IL-6 using a chemiluminescent multiplex ELISA-based assay according to the manufacturer's instructions (Quansys Biosciences Q-Plex). TMArray, Logan, UT).

[0373] <Hemagglutination inhibition titer> For hemagglutination inhibition (HI) titers, serum was pretreated with receptor-destroying enzyme II (RDE; Vibrio cholerae neuraminidase; YCC-340; Accurate Chemical and Scientific, Westbury, NY) to remove nonspecific inhibitors by diluting 1 part serum with 3 parts enzyme and incubating at 37°C for 18 h. RDE was subsequently inactivated by heating at 56°C for 45 min. Serum samples were diluted in PBS in 96-well round-bottom microtiter plates (Fisher Scientific, Pittsburg, PA). After dilution of serum, 8 HA units / well of influenza A / CA / 04 / 2009 (H1N1pdm) or influenza A / Victoria / 3 / 75 (H3N2) virus plus turkey red blood cells (Lampire Biological Laboratories, Pipersville, PA) were added (50 μL per well), mixed briefly, and incubated at room temperature for 60 min. The HI titer of a serum sample is expressed as the reciprocal of the highest serum dilution at which hemagglutination was completely inhibited.

[0374] <Virus neutralization titer> For anti-influenza virus neutralizing antibody assays, MDCK cells were cultured at 1 × 10 per well in MEM containing 5% FBS (Hyclone, Logan, UT) for 24 h prior to use. 4Cells were seeded into 96-well plates. Serial two-fold dilutions of serum samples were prepared in serum-free medium containing 10 units / mL trypsin and 1 μg / mL EDTA, starting at a 1:32 dilution and ending at 1:4096. Each serum dilution was mixed 1:1 (0.1 mL) with serum-free medium (with trypsin and EDTA) containing approximately 100 CCID50 / well H1N1pdm or influenza A / Victoria / 3 / 75 (H3N2) virus. After 1 h incubation at room temperature, the serum-influenza virus mixture (0.2 mL) was transferred to wells containing MDCK cells and incubated for 3 days. Anti-influenza virus neutralizing antibodies were measured as cytopathic effect (CPE) inhibition. CPE was scored from duplicate samples by examining the MDCK cell monolayer under a light microscope on day 3 post-infection.

[0375] <Statistical analysis> Data obtained in in vivo studies are presented as mean values ​​with standard error of the mean (SEM), and in vitro data are presented as mean values ​​with standard deviation (SD). For in vitro data, two-tailed Welch t-test was used to compare two groups. For antigen-specific antibodies, flow cytometry analysis for immune cell populations, Kruskal-Wallis test with Dunn's post-hoc test was applied for BCR-seq, TCR-seq, lung virus titer, HI endpoint titer and VN endpoint titer. Correlation between lung virus titer and cytokine / chemokine levels was analyzed using Spearman rank correlation test. For body weight, area under the curve was calculated for each mouse and one-way ANOVA was used for statistical analysis. Log-rank (Mantel-Cox) test was used to test for significant differences between Kaplan-Meier survival curves. Prism 5 software (GraphPad Software, San Diego, CA) was used. P values ​​less than 0.05 were considered statistically significant.

[0376] [Table 8]

[0377]

Table 9

[0378]

Table 10

[0379] <Result> <2B182C was obtained from the study of the structure-activity relationship of 1Z105> To improve the potency against the small molecule pyrimidoindole TLR4 ligand, 1Z105, a structure-activity relationship analysis was performed (which chemists will supplement). A total of 56 compounds were synthesized and screened by human and mouse HEK TLR4 reporter cells (HEK-Blue mTLR4 and hTLR4, respectively). Among these SAR compounds, 2B182C was discovered as a derivative with higher TLR4-stimulating potency in both mouse and human reporter cells. The EC 50 of 2B182C was examined using HEK TLR4 reporter cells and compared with the EC50 of 1Z105 (Figure 21B). The EC50 of 2B182C in mouse and human TLR4 reporter cells increased 5.8-fold and 870-fold, respectively, compared to the EC50 of 1Z105. These data indicate that the SAR study successfully yielded derivatives with higher TLR4-stimulating potency, particularly human TLR4 potency.

[0380] <The TLR4 agonist 2B182c enhanced the production of antigen-specific IgG1> The TLR4 agonist 1Z105 induced Th2-mediated IgG1 production, and the TLR7 agonist 1V270 enhanced Th1 cell immunity against influenza virus (Goff et al., J. Virol., 89:3221 (2015); Goff et al., J. Virol., 91:e01050 (2017)). We hypothesized that combining with 1V270 could improve the efficacy of the TLR4 agonist 2B182C as an influenza vaccine adjuvant. Therefore, we investigated whether 2B182C with 1V270 would improve its adjuvantity in vivo compared to a combo adjuvant using 1Z105 and 1V270.

[0381] To develop an effective combined vaccine adjuvant, the potency and optimal dose of 1Z105 and 2B182C as single agents were compared using inactivated influenza A virus [A / California / 04 / 2009(H1N1)pdm09] (IIAV) as antigen. BALB / c mice were immunized with IIAV mixed with TLR4 agonist, 1Z105 or 2B182C on days 0 and 21 and bled on day 28 (Figure 22A). Sera were evaluated by ELISA for antibodies (IgM, IgG1, and IgG2a) against two glycoproteins on the surface of the virus, hemagglutinin (HA) and neuraminidase (NA). 1Z105 and 2B182C were dissolved in DMSO, and the final concentration of DMSO was 10%. The results showed that 2B182C with a higher dose of 200 nmol / injection significantly increased IgG1 antibodies against both HA and NA (Figure 22B). Interestingly, 2B182C enhanced anti-NA specific IgG2a, but 1Z105 did not (Figure 12C). Anti-HA IgM levels were only slightly increased by 2B182C (Figure 24A).

[0382] Combination of 2B182C and TLR7 agonist 1V270 increased both antigen-specific IgG1 and IgG2a. Next, the co-adjuvant effect of these TLR4 agonists on antibody production was analyzed when combined with the TLR7 agonist 1V270 at a dose of 1 nmol / injection. 1V270 was reported to induce IgG2a production, which enhances Th1 immune responses (Goff et al., 2017). The results showed that 1V270 alone induced only anti-HA IgG2a production, but when combined with 2B182C, IgG1 and IgG2a antibodies against both HA and NA were significantly induced. This suggests that these compounds may act in an additive manner (Figures 23A and 23B). On the other hand, 1Z105 did not enhance IgG2a production induced by 1V270. Animals in the 1V270+2B182C group produced higher amounts of both IgG1 and IgG2a, suggesting that the immune balance tended toward Th1-mediated IgG2a production, and that this treatment contributed to enhancing the Th1 immune response (Figure 23C). The combination of 1V270 and 2B182C showed a moderate effect on anti-HA IgM production (Figure 24B).

[0383] In summary, the combination of 200 nmol / injection of 2B182C and 1 nmol / injection of 1V270 induced the highest amount of antigen-specific IgG1 and IgG2a and Th1-biased immune responses, which are desirable for protection against influenza virus infection, and was therefore selected for further formulation.

[0384] Liposomal formulation improved 2B182C reducing cytotoxicity Considering the above results, a 1 / 200 1V270 / 2B182C ratio (TLR4 / TLR7) was used [1 nmol / injection (20 μmM) of 1V270 and 200 nmol / injection (4 mM) of 2B182C]. Tailoring the formulation of compounds to avoid undesired cytotoxicity and reactogenicity while maintaining the response to the vaccine can be important in the development of vaccine adjuvants. Thus, 1V270 and 2B182c were formulated in liposomes by Inimmune Corp (Missoula, MT). The activity of the formulated compounds was tested in mouse primary BMDCs. These formulated compounds maintained similar levels of IL-12 secretion as the DMSO-formulated compounds (Figure 25A). Cytotoxicity induced by DMSO-2B182C or DMSO-1V270+2B182C was significantly improved by the liposomal formulation (Figure 25B). Histological analysis by H&E staining of muscles at the injection site is shown in Figure 25C. Multiple cytokine / chemokine analysis of serum after compound administration is shown in Figure 25D.

[0385] Liposomes 1V270 and 2B182C synergistically enhanced the production of anti-HA and anti-NA IgG1, as well as IgG2a. The adjuvanticity of the compounds in vivo was evaluated using the prime-boost regimen described in Figure 22A. Serum collected on day 28 was evaluated for antigen-specific antibodies by ELISA. The results showed that Lipo-2B182C induced higher levels of IgG1, which was consistent with DMSO-2B182C (Figure 26A). Unlike DMSO-1V270, Lipo-1V270 alone did not promote IgG2a production (Figure 26B). Despite these minimal effects on IgG2a by each agonist, when the two adjuvants were combined, the production of antigen-specific antibodies was synergistically enhanced (Figure 26B). Meanwhile, the total IgG levels induced by the liposomal vehicle, 1V270, 2B182C and 1V270+2B182C were comparable (Figure 26C). Antigen-specific IgM levels were not affected by any of the treatments (Figure 27). Consistent with the trends observed with the DMSO formulation, the liposome complex adjuvant induced a Th1 biased immune balance (FIG. 26D).

[0386] <Formulated 1V270 plus 2B182C enhanced antibody secretion responses> To investigate whether the formulated adjuvants induce B cell activation that promotes antigen-specific antibody secretion, lymphocytes in inguinal lymph nodes were examined for Tfh cells, GC B cells, plasmablasts, and plasma cells using flow cytometry. Using the above immunization protocol, lymphocytes in inguinal lymph nodes were collected on day 28 and analyzed by flow cytometry (Figures S28A and 28B). As a result, the percentage of Tfh cells, identified as CD3+ CD4+ PD-1+ CXCR5+ cells, was significantly increased by Lipo-1V270+2B182C (Figures 28B and 29). Furthermore, the composite adjuvant increased the percentage of GC B cells (CD3- CD19+ CD95+ GL7+). An increase in plasmablasts and plasma cells was observed in mice vaccinated with Lipo-1V270+2B182C. These results suggest that the combined adjuvant enhances the GC response compared with the single agents.

[0387] <Increased BCR diversity and TCR clonality by combo adjuvant using 1V270 and 2B182C> To examine whether the composite adjuvant affects BCR diversity, next generation sequencing analysis was performed on the IGH gene (by Repertoire Genesis Inc, Osaka, Japan). A prime boost IIAV model was used, and lymphocytes from inguinal lymph nodes were collected on day 28 (Figure 30A). BCR sequence analysis showed that BCR diversity, normalized to the total number of reads as indicated by Pielou's index, was significantly increased by Lipo-1V270+2B182C (Figure 30A). A similar analysis was performed to analyze the clonotype of the IgG gene, comparing the IGH clones between the two mice in the group to determine whether there were shared clones, and calculating the Jaccard index; Jaccard index; J(A,B)=(A∩B) / (A∪B) (Figure 30B). The Jaccard index of IGH, IGHG1 and IGHG2A was significantly increased by Lipo-1V270+2B182C, indicating that the clones shared between the two mice in this group were increased. Furthermore, in the Lipo-1V270+2B182c group, 6 clones (0.03%) were shared between three mice. These results suggest that the liposomal complex adjuvant increased the BCR diversity in total IGH and IGHG2A. This is consistent with the higher IgG2a levels after immunization with the complex adjuvant. The common clones detected in the complex adjuvant immunized group may recognize the dominant epitope of the antigen. TCR sequencing was performed to examine whether the formulated adjuvant contributed to the increase in f TCR clonality to the antigen. As expected, the complex adjuvant and Lipo-2B182C increased the clonality of TCRα and TCRβ (Figure 30C). Taken together, Lipo-1V270+2B182C animals showed higher BCR diversity and TCR clonality, which may support the data that Th1 responses are enhanced by the complex adjuvant.

[0388] <Lip-2B182C and Lip-1V270+2B182C protect mice against homologous influenza virus.> The composite adjuvants induced Th1-biased immune responses with diverse BCR and high clonality of TCR. To test whether this diversity could be indicative of immune responses to influenza virus, formulated 1V270 and 2B182c were tested in homologous and heterologous influenza virus challenge models. Balb / c mice vaccinated with IIAV plus liposomal 1V270, 2B182C or 1V270+2B182C were challenged intranasally with homologous (H1N1) influenza virus 21 days after vaccination (single dose). Mouse weight and survival were monitored for an additional 21 days (Figure 31A). Lipo-2B182C and lipo-1V270+2B182C significantly suppressed weight loss after viral infection (Figure 31B). Furthermore, against the homologous influenza virus, Lipo-1V270 showed 90% protection, while Lipo-2B182C and Lipo-1V270+2B182C completely protected mice (Figure 31C). To evaluate whether mouse survival correlated with virus titer in the lungs, bronchoalveolar lavage was performed for virus titer in the lavage fluid. The results showed that Lipo-1V270+2B182C effectively suppressed virus titer on day 6 (Figure 31D). In humans, upregulation of cytokines and chemokines in airway epithelial cells (e.g., MCP-1, IL-6, etc.) correlates with lethal lung injury and pneumonia (Gurczynski et al., Mucosal Immun., 12:518 (2019); Zhou et al., Nature, 499:500 (2013)). Therefore, we evaluated the levels of proinflammatory cytokines (IL-6) and chemokines (MCP-1) in lung fluids using Quansys multiplex ELISA. The results showed that the composite liposomal adjuvant significantly suppressed the production of both MCP-1 and IL-6 (Figure 31E). The levels of proinflammatory cytokines were correlated with lung viral titers [MCP-1 (P<0.0001, Spearman r=0.83), IL-6 (P<0.0001, Spearman r=0.79)] (Figure 31F). This trend was further enhanced in the Lipo-1V270+2B182C group.These results suggested that the composite adjuvant reduced lung damage by inhibiting viral entry and proliferation after infection. To assess whether protection was related to hemagglutination inhibition titers (HI) and virus neutralization titers (VN), sera were collected 21 days after immunization and examined for HI and VN (Figure 31A). Increased HI titers compared to the non-immunized group were observed in 19 out of 20 mice treated with lipo-1V270, lipo-2B182C, and lipo-1V270+2B182C (Figure 31G). Furthermore, lipo-2B182c and lipo-1V270+2B182C induced significantly higher VN compared to the liposome control (Figure 31H). VN titers were negatively correlated with lung viral titers (P<0.01, Spearman r=-0.59, Figure 27I). Protection against heterologous influenza virus A / Victoria3 / 75 (H3N2) was evaluated using the same protocol as in the homologous challenge experiment (Figure 31A). There were no significant differences in weight loss, survival rate and lung viral titers compared to the liposome control group (Figure 32A-C). In summary, the formulated conjugate adjuvant was insufficient for heterologous protection but showed significant protection against the homologous H1N1 virus without adverse inflammatory effects.

[0389] [Table 11]

[0390] Example 5 Liposome coencapsulation of 1V270 (TLR7 ligand) and 2B182C (TLR4 ligand) broadens antibody epitopes. A universal vaccine for influenza virus infection requires the induction of antibodies that recognize broad epitopes on the major antigenic molecules, hemagglutinin (HA) and neuraminidase (NA). Therefore, we investigated the epitope spread and cross-reactivity of antibodies induced by the composite adjuvants (1V270 and 2B182C). BALB / c mice (n = 5–10) were immunized with inactivated virus mixed with liposomal formulations of 1V270 (Lipo-1V270), 2B182C (Lipo-2B182C), coencapsulated liposomes 1V270 + 2B182C [Lipo-(1V270 + 2B182C)], and Lipo-1V270 and Lipo-2B182C in separate liposomes. Blank liposomes were used as a control, immunizations were performed on days 0 (prime) and 21 (boost), and serum was collected on day 28.

[0391] Epitope spread was assessed by HA peptide ELISA. Overlapping HA peptide arrays (139 peptides) of influenza A(H1N1)pdm09 virus were obtained from the BEI resource. Pooled peptides consisting of five consecutive peptides (total of 28 pools) were arrayed on an ELISA plate. Sera diluted 1:200 were tested for reactivity to each peptide pool by OD405-570. The OD of each serum was plotted on a heat map (Figure 38A) and the average OD of individual animals was compared. Sera from mice vaccinated with co-encapsulated liposomal 1V270+2B182C [Lipo-(1V270+2B182C)] showed significantly higher OD compared to single ligand or mixture liposomal formulations (Figure 38B). These data indicate that Lipo-(1V270+2B182C) induced an antibody response that recognizes a broad range of HA epitopes.

[0392] To investigate whether the recognition of a broad range of HA epitopes induced by Lipo-(1V270+2B182C) is associated with cross-protection against different influenza virus infection subtypes, we verified the cross-reactivity of antibodies against various subtypes of HA and NA by ELISA (Figures 39 and 40). HA and NA subtypes belonging to different phylogenetic distances. Geometric mean titers (GMTs) of IgG from mice immunized with coencapsulated Lipo-(1V270+2B182C) showed high reactivity not only with HA from group 1 (H1, H11, H12) but also with HA from group 2 (H3 and H7) compared with liposome single ligand or two separate liposomes added together. A broad reactivity was also observed against different subtypes of NA. In summary, antibodies produced in animals vaccinated with IIAV and Lipo-(1V270+2B182C) showed high cross-reactivity against different subtypes of HA and NA.

[0393] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described with reference to certain preferred embodiments thereof, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is capable of additional embodiments, and that the specific details described herein may be modified considerably without departing from the underlying principles of the invention. [Brief description of the drawings]

[0394] [Figure 1] Exemplary liposomal formulations. [Diagram 2]In vitro immunostimulatory activity of 1V270 (1 μM), 2B182C (200 μM), or a combination of 1V270 (1 μM) and 2B182C (200 mM) in DMSO or liposomal formulation. Mouse bone marrow-derived dendritic cells from wild-type C57BL / 6 mice were incubated with 1V270 (1 μM), 2B182C (200 μM), or a combination of 1V270 (1 μM) and 2B182C (200 mM) in DMSO or liposomal formulation for 18 h. Release of IL-6 in culture supernatants was measured by ELISA. [Diagram 3] Liposomal formulations reduce TLR4-independent cytotoxicity. Mouse bone marrow-derived dendritic cells from wild-type C57BL / 6 mice or TLR4-deficient mice (C57BL / 6 background) were incubated with 1V270 (1 μM), 2B182C (200 μM), or a combination of 1V270 (1 μM) and 2B182C (200 mM) in DMSO or liposomal formulations for 18 h. Cell viability was assessed by MTT assay. [Figure 4] Exemplary Experimental Protocol. [Diagram 5] Anti-HA IgG1 and IgG2a levels against formulations. [Figure 6] IgG2a / IgG1 ratio for formulation. [Figure 7] Gating strategy for GC cells and plasmablasts. [Figure 8] Cell types induced by administration of 1V270 and / or 2B182c or AddaVax. The combination of formulated 1V270 and 2B182c significantly increased the numbers of GC B cells and plasmablasts. [Figure 9] Anti-HA IgG levels measured by ELISA or as BCR-seq induced by administration of 1V270 and / or 2B182c or AddaVax. IgG2a production was strongly increased by the combination treatment and Th1 responses were induced by formulated 2B182c. [Figure 10] BCR diversity was increased by the combination treatment. [Figure 11] TCR clonality following administration of antigen with V270 and / or 2B182c or Addavax. TCR clonality was increased following 2B182c treatment and Addavax. [Figure 12] BCR diversity and TCR clonality following administration of 1V270 and / or 2B182c or Addavax. BCR diversity was increased by the combination treatment, and TCR clonality was increased following 2B182c treatment and Addavax. [Figure 13] Clonal similarity. [Figure 14] Shared clones. [Figure 15] Cluster analysis. [Figure 16] The number of clusters with similar sequences as known antibodies against influenza. [Figure 17] Analysis of cytotoxicity and IL-12 secretion. The liposomal adjuvants induced IL-12 secretion in BMDCs with lower cytotoxicity. [Figure 18] Anti-NA IgG1 and IgG2a analysis following administration of 1V270 and / or 2B182c (unformulated and formulated) or AddaVax. [Figure 19] Liposomal formulations of 2B182c and 1V270 bias the immune response towards a Th1 response. (A) BALB / c mice were immunized with inactivated Cal 2009 H1N1 influenza virus (10 μg / injection) mixed with TLR4 and / or TLR7 ligands in DMSO (D) or liposomal (L) formulations on days 0 and 28. Serum was collected on day 28. HA- or NA-specific IgG1 and IgG2a were determined by ELISA. (B) Th1 / TH2 balance was assessed by IgG2a / IgG1 ratio. *P<0.05, **P<0.001 by Mann-Whitney test. [Figure 20]Combination adjuvant treatment with liposomal 2B182c and liposomal 1V270 increased the numbers of germinal center B cells and plasmablasts in the draining lymph nodes. (A) Experimental protocol. (B) Gating strategy for flow cytometry data. (FIG. 20C) The total numbers of B cells, germinal center B cells (CD3- CD19+ CD95+ GL7+), and plasmablasts (CD3- CD19+ CD138+) were calculated. BL; blank liposome. *; p<0.05, **; p<0.01, ***; p<0.001 compared to antigen+BL by Kruskal-Wallis test with Dunn's post-hoc test. [Figure 21] 2B182C is effective against both human (A) and mouse (B) TLR4 at lower concentrations. HEK TLR reporter cells (HEK-Blue™hTLR4 and HEK-Blue™mTLR4) were treated with compounds 1Z105 and 2B182C (2-fold serial dilutions from 10 μM) for 20 h. NF-kB-induced NF-kB SEAP levels in culture supernatants were assessed according to the manufacturer's protocol. [Figure 22] 2B182c at 200 nmol / injection induced higher levels of antigen-specific IgG1 and anti-NA IgG2a. (A) Experimental protocol for comparing the two TLR agonists 1Z105 and 2B182C. BALB / c mice (n=5 / group) were immunized with IIAV (10 μg / injection) plus the TLR4 agonists 1Z105 or 2B182C (40 and 200 nmol / injection) intramuscularly in both hind legs on days 0 and 21. Blood was collected on day 28 and sera were assessed for antibodies against hemagglutinin (HA) and neuraminidase (NA) by ELISA. 10% DMSO was used as vehicle. (B) Anti-HA IgG1 and anti-NA IgG1 antibodies. (C) Anti-HA IgG2a and anti-NA IgG2a antibodies. In each box plot, the line within the box represents the median, the boundaries are upper and lower quartiles, and the bars indicate the minimum and maximum values. *P<0.05, **P<0.001 compared with antigen+vehicle by Kruskal-Wallis test with Dunn's post-hoc test. [Diagram 23] Combination of 2B182C with the TLR7 agonist 1V270 increased both antigen-specific IgG1 and IgG2a. (A-C) BALB / C mice (n=5-6) were immunized with IIAV and adjuvants as shown in Fig. 2A. AddaVaxTM, a formulation similar to MF59, was used as a positive control. Anti-HA IgG1 and anti-NA IgG1 production (A), anti-HA IgG2a and anti-NA IgG2a production (B) were determined by ELISA. In each box plot, the line within the box represents the median, the boundaries are the upper and lower quartiles, and the bars indicate the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001 by Kruskal-Wallis test with Dunn's post-hoc test. Comparisons were made between no antigen and the four groups except AddaVax (all pairs). (C) Anti-HA IgG1 and IgG2a levels (normalized to vehicle) induced by all combination treatments are shown by the average of 5-11 mice / group. Each dot represents an individual animal. The solid black line indicates IgG2a / IgG1=1. All animals immunized with the combination of 1V270 and 2B182C were distributed above IgG2a / IgG1=1. This suggests that the immune balance in these mice was biased towards Th1 immune responses. [Figure 24] Antigen-specific IgM production on day 28. (A and B) BALB / c mice (n=5-6) were immunized with IIAV (10 μg / injection) and adjuvants as indicated in Figure 2A. Antigen-specific IgM levels were measured by ELISA. (A) Anti-HA IgM and anti-NA IgM production induced by TLR4 agonists 1Z105 or 2B182C (40 and 200 nmol / injection). (B) The combination of TLR7 agonist 1V270 (1 nmol / injection) with TLR4 agonists 1Z105 or 2B182C (200 nmol / injection) had a slight effect on antigen-specific IgM induction. *P<0.05 by Kruskal-Wallis test with Dunn's post-hoc test. [Fig. 25A-25B]Liposomal 1V270 and liposomal 2B182C induced comparable levels of IL-12 release with lower toxicity. (A) IL-12 secretion levels. (B) % viability. Mouse primary BMDCs were treated with 1V270 (0.0625uM) and 2B182C (12.5uM). The 1V270 / 2B182c ratio was kept at 1:200 and judged as the best ratio in Figure 3. After overnight incubation, IL-12 levels in the culture supernatants were examined by ELISA and cell viability was assessed by MTT assay. *P<0.05, **P<0.01 for DMSO formulation (D) vs. liposomal formulation (L) for each compound by one-tailed unpaired t-test with Welch correction. [Figure 25C] Histological analysis of local immune cell infiltration after injection of combined adjuvants. Liposomal formulations of 1V270 (1 nmol / injection), 2B182C (200 nmol / injection), or a combination of 1V270 (1 nmol / injection) and 2B182C (200 nmol / injection) were injected intramuscularly into BALB / c mice. Tissues were harvested, fixed, and embedded in paraffin blocks. 10 μm sections were stained with H&E. Low and high magnification images were obtained using 20× and 40× objectives, respectively. Scale bars in low and high magnification images indicate 50 μm and 20 μm, respectively. [Figure 25D]Vehicle, 1V270, 2B182C, 1V270+2B182C were injected intramuscularly into BALB / c mice (n=5 / group) with DMSO or liposomal formulations (1nmol / injection of 1V270 and 200nmol / injection of 2B182C in a volume of 50μL). AddaVax™ (25μL / injection) was used as a positive control. Serum was collected 2 and 24 hours later and IL-12p40, TNF, and KC secretion were examined by Luminex multiplex cytokine assay (A). Data are presented as mean±SEM. *P<0.05, **P<0.01 by two-tailed Mann-Whitney U test. Kruskal-Wallis test with Dunn's post-hoc test comparing the four groups (vehicle, 1V270, 2B182C, 1V270+2B182C in the same formulation) +P<0.05, ++P<0.01. [Figure 26]Liposomal 1V270 and liposomal 2B182C synergistically enhanced anti-HA IgG1 and IgG2a production, and anti-NA IgG1 and IgG2a production. (AC) As shown in Figure 22A, BALB / c mice (n=5 / group) were immunized by intramuscular injection with IIAV (10 μg / injection) and formulated adjuvants on days 0 and 21. Liposomal TLR7 agonist 1V270 (Lipo-1V270, 1 nmol / injection), liposomal TLR4 agonist 2B182C (Lipo-2B182C, 200 nmol / injection), and liposomal complex adjuvant of 1V270 and 2B182C (Lipo-1V270+2B182C, 1 nmol / injection+200 nmol / injection) were injected. Vehicle was 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol (DOPC / Chol, control liposomes). AddaVax™ was used as a positive control. Serum was collected on day 28 and HA or NA specific IgG1, IgG2a, and total IgG were determined by ELISA. *P<0.05 and **P<0.01 by Kruskal-Wallis test with Dunn's post-hoc test. All four groups were compared except for no antigen and AddaVax (all pairs). Data are representative of two independent experiments with similar results. [Figure 27]Antigen-specific IgM levels induced by formulated adjuvants. BALB / c mice (n=5 / group) were immunized intramuscularly with IIAV (10 μg / injection) with formulated adjuvants as shown in FIG. 2A on days 0 and 21. The liposomal TLR7 agonist 1V270 (Lipo-1V270, 1 nmol / injection), the liposomal TLR4 agonist 2B182C (Lipo-2B182C, 200 nmol / injection), and the composite liposomal adjuvant of 1V270 and 2B182C (Lipo-1V270+2B182C, 1 nmol / injection+200 nmol / injection) were injected. The vehicle was 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol (DOPC / Chol, control liposome). AddaVax™ was used as a positive control. Serum was collected on day 28 and tested for IgM specific for HA or NA. *P<0.05 by Kruskal-Wallis test with Dunn's post-hoc test. Comparisons were made between no antigen and the four treatments other than AddaVax (all pairs). Data are representative of two independent experiments with similar results. [Figure 28] Formulated conjugate adjuvants increased Tfh and antibody secreting cells. (A) BALB / c mice (n=4-5 / group) were vaccinated with IIAV (10 μg / injection) with 1V270 (1 nmol / injection) and / or 2B182C (200 nmol / injection) in a total volume of 50 μL on days 0 and 21. On day 28, lymphocytes in inguinal lymph nodes were harvested for FACS analysis. Gating strategies for Tfh cells (CD3+ CD4+ PD-1+ CXCR5+), GC B cells (CD3- CD19+ CD95+ GL7+), plasmablasts (CD3- CD19+ CD138+), and plasma cells (CD3- CD19- CD138+) are shown. (B) % Tfh cells, GC B cells, plasmablasts, and plasma cells among live cells. Bars indicate mean ± SEM. *P<0.05 and **P<0.01 by Kruskal-Wallis test with Dunn's post hoc test. Four conditions were compared except AddaVax (all pairs). [Figure 29]The formulated conjugate adjuvant increased Tfh and antibody-secreting cells. BALB / c mice (n=4-5 / group) were vaccinated with IIAV (10 μg / injection) with 1V270 (1 nmol / injection) and / or 2B182C (200 nmol / injection) in a total volume of 50 μL on days 0 and 21. On day 28, lymphocytes in the inguinal lymph nodes were harvested for FACS analysis (Figure 5A). The gating strategy for Tfh cells (CD3+ CD4+ PD-1+ CXCR5+), GC B cells (CD3- CD19+ CD95+ GL7+), plasmablasts (CD3- CD19+ CD138+), and plasma cells (CD3- CD19- CD138+) is shown in Figure 5B. (A) Numbers of Tfh cells, GC B cells, plasmablasts, and plasma cells. (B) Total number of cells. Bars represent the mean±SEM. *P<0.05 and **P<0.01 (all pairs) by Kruskal-Wallis test with Dunn's post-hoc test. [Diagram 30] Formulated combination of 1V270 and 2B182C. (A and B) BALB / c mice were vaccinated with IIAV with formulated adjuvant on days 0 and 21, and inguinal lymph nodes were harvested on day 28 for BCR repertoire analysis. (A) BCR diversity for total IGH, IGHG1, and IGHG2A. (B) Similarity analysis. Jaccard index is shown. (C) TCR clonality as indicated by "1-pielou index" for TCRα and TCRβ. Bars indicate mean ± SEM. *P<0.05 and **P<0.01 (compared to liposomes) by Kruskal-Wallis test with Dunn's post-hoc test. [Diagram 31]Lipo-2B182C and Lipo-1V270+2B182C protect mice against homologous influenza virus. (A) Experimental schedule of homologous influenza virus challenge. (B) Mean body weight change is shown by % initial body weight. *P<0.05, **P<0.01 by one-way ANOVA with Dunnett's post-hoc test. (C) Survival rate of mice after challenge with homologous virus (H1N1pdm). Kaplan-Meier curves with log-rank test are shown. Lung virus titers (D) and cytokine levels in lung fluid (E) were assessed. Lung lavage was performed on days 3 and 6. **P<0.01 (compared to liposomes) by Kruskal-Wallis test with Dunn's post-hoc test. (F) Relationship between lung virus titers and pro-inflammatory cytokines MCP-1 (left) and IL-6 (right). Spearman rank correlation test (MCP-1; **P<0.0001, Spearman r=0.83; IL-6; ***P<0.0001, Spearman r=0.79). HI titers (G) and VN titers against the same virus (H). *P<0.01 and ***P<0.001 (all pairs) by Kruskal-Wallis test with Dunn's post-hoc test. (I) Relationship between VN titers and lung virus titers. Each dot represents the VN titer and lung virus titer in the same animal. **P<0.01, Spearman rank correlation test, Spearman r=-0.59. [Diagram 32] Heterologous challenge with H3N2 virus. BALB / c mice were immunized with IIAV(H1N1) plus formulated adjuvant and challenged intranasally with the heterologous virus A / Victoria3 / 75(H3N2) as described in FIG. 31A. (A) Weight loss was monitored. No significance was detected by one-way ANOVA. (B) Survival rate of mice after heterologous virus challenge. Kaplan-Meier curves with log-rank test (ns) are shown. (C) Lung virus titers at days 3 and 6. No significance was detected by Kruskal-Wallis test. [Diagram 33]A and E) Protocol. BC and FG) Body weight and survival over time following infection with A / PuertoRico / 8 / 1934 or B / Florida / 04 / in mice treated with 1V270 and / or 2B182C or AddaVax. D) IgG2a / IgG1 ratio in mice treated with 1V270 and / or 2B182c or AddaVax. [Diagram 34] A) Anti-HA IgG1, anti-HA IgG2a, and anti-HA IgM in mice treated with 1V270, 1Z105, 2B182c, or AddaVax. B) Anti-NA IgG1, anti-NA IgG2a, and anti-NA IgM in mice treated with 1V270, 1Z105, 2B182c, or AddaVax. [Diagram 35] A and B) Anti-HA and anti-NA IgG1, CD) Anti-HA and anti-NA IgG2a, and EF) Anti-HA and anti-NA IgM in mice treated with 1V270, 1Z105, 2B182c, or AddaVax. B) Anti-NA IgG1, anti-NA IgG2a, and anti-NA IgM in mice treated with 1V270, 1Z105, 2B182c, or AddaVax. [Diagram 36] Anti-HA IgG2a and IgG1 in mice treated with different doses of 1V270, 1Z105, 2B182c, or their combinations. [Figure 37] Schematic diagrams of various liposomes and exemplary protocols. [Figure 38]ELISA using peptide array of HA of A / California / 04 / 2009(H1N1)pdm. BALB / c mice (n=5-10) were immunized with IIAV plus Lipo-Veh (blank liposomes), Lipo-1V270, Lipo-2B182C, Lipo-(1V270+2B182C) (coencapsulated combination), or (Lipo-1V270)+(Lipo-2B182C) (mixed combination) on days 0 and 21, and bled on day 28. Peptide array of HA of A / California / 04 / 2009(H1N1)pdm (NR-15433) was obtained from BEI Resources. Five groups of peptides were pooled to generate 28 peptide pools. (A) Heat map of OD405-570nm of ELISA results. Each row and column represents a peptide pool and a mouse, respectively. (B) Statistical analysis was performed on the average of 28 peptide pools in each mouse. **P<0.01, ***P<0.0001, Kruskal-Wallis test with Dunn's post-hoc test. +P<0.05, Mann-Whitney test. [Figure 39]ELISA for antibody cross-reactivity. BALB / c mice (n=5 / group) were immunized with IIAV plus Lipo-Veh, Lipo-1V270, Lipo-2B182C, Lipo-(1V270+2B182C), or (Lipo-1V270)+(Lipo-2B182C) on days 0 and 21 and bled on day 28. Sera were serially diluted (1:100 to 1:409600) and assessed by ELISA for total IgG levels against Puerto RicoH1N1, H11N9, H12N5, H7N7, and H3N2 HAs, and H5N1, H10N8, H3N2, and H7N7 NAs. (A) Phylogenetic relationships of the HAs of influenza A viruses used in this study. The amino acid sequences of proteins used in ELISA were aligned by the MUSCLE algorithm using the Influenza Research Database (https: / / www.fludb.org / brc / home.spg?decorator=influenza). Phylogenetic trees were constructed by the neighbor-joining method using MEGAX software (https: / / www.megasoftware.net / ). (B) Total IgG titer curves against HA of H1N1, H11N9, H12N5, H3N2, and H7N7. (C) Phylogenetic relationships of NA. (D) Total IgG titer curves against NA of H5N1, H10N8, H3N2, and H7N7. Sera were diluted 1:4 starting from 100-fold to 409600-fold, and total IgG levels were assessed by ELISA. Data shown are mean ± SEM. [Diagram 40]Lipo-(1V270+2B182C) induced cross-reactive antibodies. (A and B) BALB / c (n=5 / group) mice were immunized with IIAV [A / California / 2009(H1N1)pdm09] plus Lipo-Veh, Lipo-1V270, Lipo-2B182C, Lipo-(1V270+2B182C), or (Lipo-1V270)+(Lipo-2B182C) on days 0 and 21 and bled on day 28. Sera were serially diluted (1:100 to 1:409600) and total IgG levels were assessed by ELISA against Puerto RicoH1N1, H11N9, H12N5, H7N7, and H3N2 HAs, and H5N1, H10N8, H3N2, and H7N7 NAs. Geometric means of total IgG titer curves of individual mice calculated using prism 5 are shown. Total IgG titer curves and phylogenetic relationships of HA proteins used in this study are shown above. *P<0.05, **P<0.01, Kruskal-Wallis test with Dunn's post hoc test. +P<0.05, ++P<0.01, Mann-Whitney test. [Diagram 41] Exemplary TLR4 and TLR7 agonists.

Claims

1. 1. Use of a liposome-containing composition in the manufacture of a medicament for enhancing an immune response in a mammal by a method comprising administering the liposome-containing composition to a mammal in need thereof, The liposome-containing composition comprises an effective amount of a TLR4 agonist and a TLR7 agonist, The TLR7 agonist is 【Chemistry 1】 and The TLR4 agonist is 【Chemistry 2】 That is, use.

2. The use of claim 1 , wherein the method comprises administering the TLR4 agonist and the TLR7 agonist simultaneously.

3. The use according to claim 1 or 2, wherein the liposome comprises PC, DOPC, or DSPC.

4. The use according to any one of claims 1 to 3, wherein the liposome comprises cholesterol.

5. The use according to any one of claims 1 to 4, wherein the method further comprises the step of administering one or more immunogens.

6. The use according to claim 5 , wherein the immunogen is a microbial immunogen.

7. The use according to claim 6, wherein the microorganism is a virus or a bacterium.

8. The use according to any one of claims 5 to 7, wherein the liposome comprises one or more immunogens.

9. The use according to any one of claims 1 to 8, wherein the mammal is a human.

10. The use according to any one of claims 1 to 9, wherein the amount of the TLR7 agonist is 0.01 to 100 nmol, 0.1 to 10 nmol, or 100 nmol to 1000 nmol.

11. The use according to any one of claims 1 to 10, wherein the amount of the TLR4 agonist is 2 to 20 μmol, 20 nmol to 2 μmol, or 2 μmol to 100 μmol.

12. 12. The use of any one of claims 1 to 11, wherein the ratio of the TLR7 agonist to the TLR4 agonist is 1:10, 1:100, 1:200, 5:20, 5:100, or 5:

200.

13. The use according to any one of claims 1 to 12, wherein the composition is administered by injection, intramuscular administration, intranasal administration or intravenous administration.

14. The use according to any one of claims 1 to 13, wherein the liposome comprises DOPC and cholesterol.

15. 10. A pharmaceutical formulation comprising a liposome, a TLR4 agonist and a TLR7 agonist as defined by claim 1.

16. The liposomes may be selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:0 PEG-2000 PE), 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-glycero-3-phosphocholine (18:1 to 12:0 NBD PC), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-glycero-3-phosphocholine (16:0 to 12:0 NBD 16. The formulation of claim 15, comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, or mixtures thereof.

17. 16. The formulation of claim 15, wherein the liposome comprises DOPC, cholesterol, or a combination thereof.

18. The formulation of any one of claims 15 to 17, wherein the amount of the TLR7 agonist is 0.01 to 100 nmol, 0.1 to 10 nmol, or 100 nmol to 1000 nmol.

19. The formulation of any one of claims 15 to 18, wherein the amount of the TLR4 agonist is from 2 nmol to 20 μmol, from 20 nmol to 2 μmol, or from 2 μmol to 100 μmol.

20. 20. The formulation of any one of claims 15 to 19, wherein the ratio of the TLR7 agonist to the TLR4 agonist is 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200.

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