TLR4 and TLR7 ligand preparations as vaccine adjuvants

Abstract

Provided are a method for enhancing the immune response of a mammal and a composition comprising liposomes, a TLR4 agonist and a TLR7 agonist.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Application No. 62 / 818,517, filed on March 14, 2019, the disclosure of which is incorporated by reference into this application.

[0003] Government Rights Statement

[0004] This invention was made with U.S. Government support under Grant HHSN272200900034C from the National Institutes of Health. The U.S. Government has certain rights in this invention. Background Art

[0005] The most effective way to protect individuals from many potential infectious diseases is through vaccination. Effective vaccination requires the use of antigens that are able to elicit an immune response in the host that is able to provide subsequent protection against the specific pathogen targeted by the vaccine. Therefore, vaccine antigens must be sufficiently immunogenic to induce a level of humoral and / or cell-mediated immune response—one that is protective in the host. Pathogens of global concern are influenza viruses. Seasonal influenza viruses cause epidemics each year, killing 250–500,000 people worldwide (WHO), with more than 80,000 deaths in the United States alone last winter. In addition, new epidemics occasionally emerge, killing millions of people—posing a very real global threat. Particularly vulnerable to these threats are high-risk groups such as the elderly, newborns, and immunocompromised individuals. Vaccination against seasonal influenza may be modestly effective if matched to the strains circulating seasonally. However, because influenza viruses are constantly changing (antigenic drift), it is difficult to predict what subtypes and strains will circulate in the next influenza season or the next pandemic, and sufficient time (about 6 months) needs to be allowed for routine vaccine production and distribution.

[0006] These conventional vaccines are typically based on antigens associated with the influenza hemagglutinin (HA) protein, specifically the head globular domain of the protein. This highly immunogenic head domain varies between different strains and subtypes of influenza virus, and therefore, immune responses against one head globular domain subtype may be limited to that specific head domain and fail to provide an appropriate immune response against strains with different head domains. Influenza HA antigens derived from the stem or rod domain of the protein are more highly conserved among virus strains and are generally much less immunogenic than the head domain antigens that predominate in conventional vaccines, and therefore the immunogenicity of these HA rod antigens needs to be increased to a level that can generate a sufficient immune response in the host to generate an immune response against multiple influenza strains. Summary of the invention

[0007] The successful use of appropriate adjuvant combination formulations in vaccines against globally important pathogens such as influenza virus could represent a major step forward in medicine, extending and enhancing the protection of individuals against the ever-changing threats of these viral pathogens.

[0008] The present invention provides a combination of a TLR4 agonist and a TLR7 agonist formulated as an adjuvant in the same liposome nanoparticle, providing several advantages over combinations in which the agonists are formulated separately and then mixed and combinations in which the agonists are not formulated. The formulated combination has a specific ratio of TLR4 to TLR7 in the nanoparticles to obtain the desired immunological activity. Based on the data obtained from the ratios of various compound combinations, each compound is formulated separately and each compound is formulated in combination. The formulated and unformulated combinations, and the mixed and combined combinations in the same particles are compared together. The results of the immune study show that certain ratios of combined compounds in the liposomes provide greater and more extensive immunological activity than the individual compounds, and the formulated combinations are better than the unformulated combinations. The antigens used are OVA and inactivated influenza virus.

[0009] As disclosed herein, 2B182C (an exemplary TLR4 agonist) and 1V270 (an exemplary TLR7 agonist) were formulated together in a formulation and immunization studies were conducted in mice. Based on the data obtained from various compound combination ratios, each compound was formulated alone and in combination. Formulated and unformulated combinations, mixed and combined combinations in the same particles were compared together. The results of the immunization studies showed that a certain ratio of the combined compounds in the liposomes provided greater and more extensive immunological activity than the individual compounds, and that the formulated combinations were better than the unformulated combinations. The antigens used were OVA and inactivated influenza virus.

[0010] In one embodiment, the present invention provides a method for enhancing a mammalian immune response, comprising administering an effective dose 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 administered simultaneously. In one embodiment, the TLR4 agonist and the TLR7 agonist are administered in a liposome formulation. In one embodiment, the TLR4 agonist and the TLR7 agonist are administered in separate liposome formulations. In one embodiment, the TLR4 agonist has a structure of formula (II). In one embodiment, the TLR7 agonist has a structure of formula (I). In one embodiment, one or more immunogens (antigens) are also administered, for example, administered simultaneously with an adjuvant, and optionally administered 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 varicella, or a bacterium. In one embodiment, the mammal is a human. In one embodiment, the dosage 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 dosage of the TLR4 agonist is about 2 to 20umol, about 20nmol to 2umol, or about 2umol to about 100umol. 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 preparation is administered by injection. In one embodiment, the liposome preparation comprises DOPC, cholesterol or a combination thereof. Also provided are pharmaceutical formulations comprising liposomes, a TLR4 agonist, and a TLR7 agonist, for example, wherein the liposomes comprise DOPC, cholesterol, or a combination thereof, wherein in one embodiment, the amount of the TLR7 agonist is approximately 0.01 to 100 nmol, approximately 0.1 to 10 nmol, or approximately 100 nmol to approximately 1000 nmol; wherein the amount of the TLR4 agonist is approximately 2 nmol to 20 umol, approximately 20 nmol to 2 umol, or approximately 2 umol to approximately 100 umol; or wherein the ratio of TLR7 to TLR4 agonist is approximately 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Exemplary liposome formulations.

[0012] Figure 2 : In vitro immunostimulatory activity of DMSO or liposomal formulations of 1V270 (1 μM), 2B182C (200 μM), or a combination of 1V270 (1 μM) and 2B182C (200 mM).

[0013] Mouse bone marrow-derived dendritic cells from wild-type C57BL / 6 mice were cultured with DMSO or liposomal formulations of 1V270 (1 μM), 2B182C (200 μM), or a combination of 1V270 (1 μM) and 2B182C (200 mM) for 18 hours. IL-6 release in the culture supernatant was measured by ELISA.

[0014] Figure 3 : Liposomal formulation alleviates TLR4-independent cytotoxicity.

[0015] Mouse bone marrow-derived dendritic cells from wild-type C57BL / 6 mice or TLR4-deficient mice (C57BL / 6 background) were cultured with DMSO or liposomal formulations of 1V270 (1 μM), 2B182C (200 μM), or a combination of 1V270 (1 μM) and 2B182C (200 mM) for 18 hours. Cell viability was assessed using the MTT assay.

[0016] Figure 4 : Exemplary experimental protocol.

[0017] Figure 5 : Anti-HA IgG1 and IgG2a levels of the preparations.

[0018] Figure 6 : IgG2a / IgG1 ratio of the preparation.

[0019] Figure 7 : Gating strategy for GC cells and plasmablasts.

[0020] Figure 8 : Cell types induced by administration of 1V270 and / or 2B182c or AddaVax. The formulated combination of 1V270 and 2B182c significantly increased the number of GC B cells and plasmablasts.

[0021] Fig. 9 : Anti-HA IgG levels induced by administration of 1V270 and / or 2B182c or AddaVax as measured by ELISA or BCR-seq. IgG2a production was significantly increased by combined treatment, and formulation of 2B182c induced a Th1 response.

[0022] Fig.10 : Combination treatment increases BCR diversity.

[0023] Fig.11 : TCR clonality after administration of antigen and 1V270 and / or 2B182c or Addavax. TCR clonality increased after 2B182c treatment and Addavax.

[0024] Fig.12 : BCR diversity and TCR clonality after administration of 1V270 and / or 2B182c or Addavax. BCR diversity was increased by the combined treatment, and TCR clonality was increased after 2B182c treatment and Addavax.

[0025] Fig.13 : Clonal similarity.

[0026] Fig.14 : Shared clones.

[0027] Fig.15 : Cluster analysis.

[0028] Fig.16 : Number of clusters with similar sequences to known anti-influenza antibodies.

[0029] Fig.17 : Cytotoxicity and IL-12 secretion analysis. Liposomal adjuvants induce IL-12 secretion in BMDCs with low cytotoxicity.

[0030] Fig.18 : Analysis of anti-NA IgG1 and IgG2a following administration of 1V270 and / or 2B182c (unformulated and formulated) or AddaVax.

[0031] Fig.19 A-19B: Liposomal formulations of 2B182c and 1V270 bias the immune response toward a Th1 response. (A) BALB / c mice were immunized on days 0 and 28 with inactivated Cal 2009 H1N1 influenza virus (10 μg / injection) mixed with DMSO (D) or liposomal (L) formulations of TLR4 ligands and / or TLR7 ligands. Serum was collected on day 28 and measured by ELISA for HA or NA-specific IgG1 and IgG2a. (B) Th1 / TH2 balance was assessed by the IgG2a / IgG1 ratio. * P<0.05, ** P<0.001, Mann-Whitney test.

[0032] Fig. 20 A-20C: The number of germinal center B cells and plasmablasts in draining lymph nodes was increased by adjuvant treatment with a combination of liposomes 2B182c and 1V270. (A) Experimental scheme. (B) Gating strategy for flow cytometry data. (C) B cells, germinal center B cells (CD3 - CD19 + CD95 + GL7 + ) and plasmablasts (CD3 - CD19 +CD138 + ) BL: blank liposomes. * ; p<0.05, ** ; p<0.01, *** ; p<0.001, Kruskal-Wallis test, Dunn's post hoc test, compared with Antigen+BL.

[0033] Fig.21 A-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 starting from 10 μM) for 20 h. NF-kB-inducible NF-kB SEAP levels in culture supernatants were assessed according to the manufacturer's protocol.

[0034] Fig. 22 A-22C: 200 nmol / injection 2B182c induces higher levels of antigen-specific IgG1 and anti-NA IgG2a. (A) Experimental scheme comparing two TLR agonists, 1Z105 and 2B182C. BALB / c mice (n=5 / group) were immunized by intramuscular injection with IIAV (10 μg / injection) and TLR4 agonists 1Z105 or 2B182C (40 and 200 nmol / injection) in both hind legs on days 0 and 21. Blood was collected on day 28, and anti-hemagglutinin (HA) antibodies and anti-neuraminidase (NA) antibodies in the serum were evaluated by ELISA. 10% DMSO was used as the solvent. (B) Anti-HA and anti-NA IgG1 antibodies. (B) Anti-HA and anti-NA IgG2a antibodies. In each box plot, the line in the box represents the median, the limits are the upper and lower quartiles, and the bars represent the minimum and maximum values. * P<0.05, ** P < 0.01, Kruskal-Wallis test, Dunn's post hoc test (vs. antigen + vehicle).

[0035] Fig.23 A-23C: The combination of 2B182C and TLR7 agonist 1V270 increases antigen-specific IgG1 and IgG2a. (AC) BALB / c mice (n=5-6) were treated with IIAV and Figure 1 Immunization with the indicated adjuvants. AddaVax TMSimilar to MF59 preparation, used as positive control. Anti-HA and anti-NA IgG1 production (A), anti-HA and anti-NA IgG2a production (B) were determined by ELISA. In each box plot, the line in the box represents the median, the limits are the upper and lower quartiles, and the bars represent the minimum and maximum values. * P<0.05, ** P<0.01, *** P<0.001, Kruskal-Wallis test, Dunn post hoc test. Four groups (all paired) were compared except no antigen and AddaVax. (C) Anti-HA IgG1 and IgG2a levels induced by all combined treatments shown in 5-11 mice / group (normalized to vehicle). Each point 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, indicating that the immune balance of these mice is biased towards a Th1 immune response.

[0036] Fig.24 A-24B: Antigen-specific IgM production on day 28. (A and B) BALB / c mice (n=5-6) were treated with IIAV (10 μg / injection) and Figure 1 Adjuvant immunization indicated. Antigen-specific IgM levels were determined by ELISA. (A) Anti-HA 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) and TLR4 agonist 1Z105 or 2B182C (200 nmol / injection) had little effect on antigen-specific IgM induction. * P < 0.05, Kruskal-Wallis test, Dunn's post hoc test.

[0037] Figures 25A-25B :Liposomes 1V270 and 2B182C induce similar levels of IL-12 release, but with lower cytotoxicity. (A) IL-12 secretion level. (B) % viability. Muse primary bone marrow derived dendritic cells (BMDC) were treated with 1V270 (0.0625uM) and 2B182C (12.5uM). The ratio of 1V270 / 2B182c was maintained at 1:200, which was Figure 3 After culturing for one night, the IL-12 level in the culture supernatant was detected by ELISA, and the cell viability was evaluated by MTT assay. * P<0.05, **P < 0.01, one-tailed unpaired t-test, Welch correction, DMSO formulation (D) versus liposomal formulation (L) of each compound.

[0038] Fig.25C : Histological analysis of local immune cell infiltration after injection of combined adjuvants. BALB / c mice were injected intramuscularly with liposomal formulations of 1V270 (1nmol / injection), 2B182C (200nmol / injection), or a combination of 1V270 (1nmol / injection) and 2B182C (200nmol / injection). Tissues were collected, fixed and embedded in paraffin blocks. 10μm sections were stained with H&E. Low and high magnifications were obtained using 20x and 40x objectives, respectively. The scale bars in low and high magnification images represent 50μm and 20μm, respectively.

[0039] Fig.25D BALB / c mice (n=5 / group) were injected intramuscularly with vehicle, 1V270, 2B182C, DMSO formulation of 1V270+2B182C, or liposome formulation [1 nmol / injection 1V270 and 200 nmol / injection 2B182C, volume 50 μL]. TM (25 μL / needle) was used as a positive control. After 2 and 24 hours, serum was collected and the secretion of IL-12p40, TNF and KC was measured by Luminex multiplex cytokine assay (A). Data shown are mean ± SEM. * P<0.05, ** P < 0.01, two-tailed Mann-Whitney U test. +P < 0.05, ++P < 0.01, Kruskal-Wallis, Dunn post hoc test, comparing 4 groups (vehicle, 1V270, 2B182C, 1V270 + 2B182C in the same formulation).

[0040] Fig.26 A-26D: Liposomes 1V270 and 2B182C synergistically enhance the production of anti-HA and anti-NA IgG1 and IgG2a. (AC) BALB / c mice (n=5 / group) were treated with IIAV (10 μg / injection) and Fig. 22Immunization was performed by intramuscular injection of the adjuvant prepared as shown in A. Liposomal TLR7 agonist 1V270 (lipo-1V270, 1nmol / injection), liposomal TLR4 agonist 2B182C (lipo-2B182C, 200nmol / injection) and liposomal combination adjuvant 1V270 and 2B182C (lipo-1V270+2B182C, 1nmo / injection+200nmo / injection) were injected. The solvent was 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol (DOPC / Chol, control liposome). AddaVax TM Serum was collected on day 28 and assayed for HA or NA-specific IgG1, IgG2a, and total IgG by ELISA. * P < 0.05 and ** P<0.01 Kruskal-Wallis test, Dunn post hoc test. Four groups (all paired) were compared except no antigen and AddaVax. Data are representative of two independent experiments with similar results.

[0041] Fig. 27 : Antigen-specific IgM levels induced by formulated adjuvants. BALB / c mice (n=5 / group) were treated with IIAV (10 μg / injection) and Figure 1 Immunization was performed by intramuscular injection of the adjuvants prepared as shown. Liposomal TLR7 agonist 1V270 (lipo-1V270, 1 nmol / injection), liposomal TLR4 agonist 2B182C (lipo-2B182C, 200 nmol / injection) and liposomal combination adjuvants 1V270 and 2B182C (lipo-1V270+2B182C, 1 nmo / injection+200 nmo / injection) were injected. The solvent was 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol (DOPC / Chol, control liposomes). AddaVax TM Serum was collected on day 28 and assayed for HA or NA-specific IgM. * P<0.05, Kruskal-Wallis test, Dunn post hoc test. Four treatments (all paired) were compared except no antigen and AddaVax. Data are representative of two independent experiments with similar results.

[0042] Fig.28A-28C: The formulated combined adjuvant increases Tfh and antibody secreting cells. (A) BALB / c mice (n=4-5 / group) were inoculated with IIAV (10 μg / pin) and 1V270 (1 nmol / pin) and / or 2B182C (200 nmol / pin) on days 0 and 21, with a total volume of 50 μL. After 28 days, lymphocytes in inguinal lymph nodes were collected for FACS analysis. 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. (B) The percentage of Tfh cells, GC B cells, plasmablasts and plasma cells in living cells. Bars represent mean ± SEM. * P<0.05, ** P < 0.01, Kruskal-Wallis test, Dunn post hoc test. Four conditions except AddaVax were compared (all pairs).

[0043] Fig.29 A-29B: The formulated combination adjuvant increased Tfh and antibody-secreting cells. (A) BALB / c mice (n=4-5 / group) were vaccinated with IIAV (10 μg / injection) and 1V270 (1 nmol / injection) and / or 2B182C (200 nmol / injection) on days 0 and 21 in a total volume of 50 μL. After 28 days, lymphocytes in the inguinal lymph nodes were collected for FACS analysis. 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+) was Fig.29 A-29B shows. (A) Number of Tfh cells, GC B cells, plasmablasts, plasma cells. (B) Total cell number. Bars represent mean ± SEM. * P<0.05, ** P < 0.01, Kruskal-Wallis test, Dunn's post hoc test (all paired).

[0044] Fig.30A-30C: a formulated combination of 1V270 and 2B182C. (A and B) BALB / c mice were vaccinated with IIAV and formulated adjuvant on days 0 and 21, and inguinal lymph nodes were harvested on day 28 for BCR library analysis. (A) BCR diversity of total IGH, IGHG1, and IGHG2A. (B) Similarity analysis. Jaccard index is shown. (C) TCR clones of TCRα and TCRβ are represented by "1-pielou index". Bars represent mean ± SEM. * P<0.05, ** P < 0.01, Kruskal-Wallis test, Dunn's post hoc test (vs. liposomes).

[0045] Fig.31 A-31I: Lipo-2B182C and lipo-1V270+2B182C protect mice from homologous influenza virus. (A) Experimental schedule for homologous influenza virus challenge. (B) Mean body weight change expressed as a percentage of initial body weight. * P<0.05, ** P < 0.01, one-way ANOVA, Dunn post hoc test. (C) Survival of mice challenged with homologous virus (H1N1pdm). Kaplan-Meier curves showing 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, Kruskal-Wallis test, Dunn post hoc test (vs. liposomes). (F) Relationship between lung virus titers and proinflammatory cytokines, MCP-1 (left) and IL-6 (right). Spearman rank correlation test, (MCP-1; ** P < 0.0001, Spearman correlation coefficient r = 0.83, IL-6; *** P<0.0001, Spearman correlation coefficient r=0.79). HI titer (G) and VN titer (H) against homologous viruses. * P<0.01, *** P < 0.001, Kruskal-Wallis test, Dunn post hoc test (all paired). (I) Relationship between VN titer and lung virus titer. Each point represents the VN titer and lung virus titer of the same animal. ** P<0.01, Spearman rank correlation test, Spearman correlation coefficient r=-0.59.

[0046] Fig.32 A-32C: Heterologous challenge with H3N2 virus. Fig.31BALB / c mice were immunized with the adjuvant and IIAV (H1N1) formulated as described in A and challenged intranasally with the heterologous virus A / Victoria3 / 75 (H3N2). (A) Weight loss was monitored. One-way ANOVA did not detect significance. (B) Survival of mice after challenge with heterologous viruses. Kaplan-Meier curves showing log-rank tests are shown. (C) Lung virus titers on days 3 and 6. Kruskal-Wallis test did not detect significance.

[0047] Fig.33 A-33G: A and E) regimen. BC and FG) body weight and

[0048] Body weight and survival rate of mice administered 1V270 and / or 2B182c or AddaVax over time after infection with A / PuertoRico / 8 / 1934 or B / Florida / 04 / . D) IgG2a / IgG1 ratio of mice administered 1V270 and / or 2B182c or AddaVax.

[0049] Fig.34 A-34B: A) Anti-HA IgG1, anti-HA IgG2a, and anti-HA IgM in mice administered with 1V270, 1Z105, 2B182c, or AddaVax. B) Anti-NA IgG1, anti-NA IgG2a, and anti-NA IgM in mice administered with 1V270, 1Z105, 2B182c, or AddaVax.

[0050] Fig.35 A-35F: A and B) anti-HA and anti-NA IgG1, C-D) anti-HA and anti-NA IgG2a, and E-F) anti-HA and anti-NA IgM in mice administered 1V270, 1Z105, 2B182c, or AddaVax. B) anti-NA IgG1, anti-NA IgG2a, and anti-NA IgM in mice administered 1V270, 1Z105, 2B182c, or AddaVax.

[0051] Fig.36 A-36B: Anti-HA IgG2a and IgG1 of mice administered different doses of 1V270, 1Z105, 2B182c or their combination.

[0052] Fig.37 : Schematic representation of various liposomes and exemplary protocols.

[0053] Fig.38A-38B: ELISA assay using HA peptide matrix of A / California / 04 / 2009(H1N1)pdm. BALB / c mice (n=5-10) were immunized with IIAV and Lipo-Veh (blank liposomes), Lipo-1V270, Lipo-2B182C, Lipo-(1V270+2B182C) (co-encapsulated combination) or (Lipo-1V270)+(Lipo-2B182C) (mixed combination) on days 0 and 21, and blood was collected on day 28. The peptide matrix of A / California / 04 / 2009(H1N1)pdm(NR-15433)HA was obtained from BEI resources. Peptides from the 5 groups were combined to generate a pool of 28 peptides. (A) OD 405-570nm Heatmap and ELISA results. Each row and column represents each peptide pool and mouse, respectively. (B) Statistical analysis of the average of 28 peptide pools from a single mouse. ** P<0.01, *** P < 0.0001, Kruskal-Wallis test, Dunn's post hoc test. +P < 0.05, Mann-Whitney test.

[0054] Fig.39A-39D: ELISA to detect cross-reactivity of antibodies. BALB / c mice (n=5 / group) were immunized with IIAV and 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 HA of Puerto Rico H1N1, H11N9, H12N5, H7N7, and H3N2, and against NA of H5N1, H10N8, H3N2, and H7N7. (A) Phylogenetic relationships of influenza A virus HA used in this study. The amino acid sequences of the proteins used in ELISA were aligned using the MUSCLE algorithm using the Influenza Research Database (https: / / www.fludb.org / brc / home.spg?decorator=influenza). The phylogenetic tree was constructed using the Neighbor-joining method using MEGAX software (https: / / www.megasoftware.net / ). (B) Total IgG titer curves of HA for H1N1, H11N9, H12N5, H3N2, and H7N7. (C) Phylogenetic relationships of each NA. (D) Total IgG titer curves of NA for H5N1, H10N8, H3N2, and H7N7. Sera were diluted 1:4 from 100 to 409,600 times, and total IgG levels were assessed by ELISA. The data shown are mean ± SEM.

[0055] Fig.40A-40B: Lipo-(1V270+2B182C) induces cross-reactive antibodies. (A and B) BALB / c (n=5 / group) mice were immunized with IIAV [A / California / 04 / 2009(H1N1)pdm09]+Lipo-Veh, Lipo-1V270, Lipo-2B182C, Lipo-(1V270+2B182C) or (Lipo-1V270)+(Lipo-2B182C) on days 0 and 21, and bled on day 28. The sera were serially diluted (1:100 to 1:409600) and total IgG levels were assessed by ELISA against HA of Puerto Rico H1N1, H11N9, H12N5, H7N7 and H3N2 and NA of H5N1, H10N8, H3N2 and H7N7. The geometric mean of the total IgG titer curves of individual mice calculated using prism5 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, Dunn's post hoc test. +P < 0.05, ++P < 0.01, Mann-Whitney test.

[0056] Fig.41 : Exemplary TLR4 and TLR7 agonists. DETAILED DESCRIPTION

[0057] The use of adjuvants in vaccines is a well-established approach to promote stronger immune responses to weakly immunogenic antigens. In addition, adjuvants can enhance and potentially extend immune responses by promoting the immunogenicity of weakly immunogenic antigens. Currently only a few adjuvants are licensed for use in vaccines (O'Hagan, et al. doi:10.1016 / j.vaccine.2015.01.088). Furthermore, most existing vaccines contain a single adjuvant, and recent evidence suggests that a single adjuvant may not be sufficient to induce a protective immune response against many emerging infectious diseases. (Underhill, doi:10.1111 / j.1600-065X.2007.00548.x).

[0058] The use of a combination of TLR agonists as adjuvants generally results in an overall enhanced immune response, but in the case of vaccines for infectious diseases such as influenza, the Th1 (cell-mediated) immune response is enhanced or biased towards the Th1 type of immune response at the expense of the Th2 (humoral or antibody) type. In fact, despite the increase in Th1 response, this sometimes results in insufficient production of protective Th2 antibodies, and for influenza infection, a certain protective antibody titer is considered to be the main factor in providing effective protection through immunization.

[0059] In the present invention, it was found that the combined ratio of TLR4 / TLR7 agonists in a single nanoparticle formulation not only enhanced the overall immune response to the antigen, but also provided sufficient protective antibody production to effectively protect mice from the attack of the deadly virus. The immune state of humans will be very different from that of mice, who are usually new to antigens such as influenza, while humans are usually exposed to influenza antigens for many years through natural exposure and vaccine exposure. The same is true for other infectious pathogens such as chickenpox (varicella zoster), which may subsequently appear in humans in the form of shingles.

[0060] It is well known that immunization with one antigen blocks a strong immune response to a second, similar antigen. This may be due to 1) epitope rejection, in which pre-existing antibodies, especially mucosal IgA, protect the vaccine from all antigen-presenting cells; 2) reduced dendritic cell (DC) access, in which memory B cells internalize the new vaccine, reducing DC access and activation and T cell immunity; 3) T cell competition, in which memory B cells are activated, consume cytokines, cofactors and capture T cells that can react with antigen-loaded DC.

[0061] The present invention overcomes these deficiencies by: 1) encapsulating the vaccine in liposome nanoparticles to preferentially deliver the vaccine to DCs; 2) activating DCs using a specific ratio of combined TLR agonists, which will increase the diversity of activated T cells against the vaccine antigens. The present invention discloses our findings that formulating a combination of a TLR4 agonist and a TLR7 agonist as an adjuvant in the same liposome nanoparticle has several advantages over a mixed combination of a single formulated agonist and an unformulated agonist. The formulated combination can have a certain ratio of TLR4 and TLR7 in the nanoparticle to provide immune activity.

[0062] Advantages of combining these ratios include: 1) enhanced activity compared to DMSO formulations, providing stronger Th1 and Th2 immune responses; 2) reduced toxicity compared to DMSO formulations; 3) protection of antigens (for vaccine use) from B cells and IgA in hyperimmune individuals, which is particularly true for mucosal influenza immunity and allows dendritic cells to present important epitopes to provide an effective protective response; and / or 4) expansion of the immune response to include immune responses to less immunogenic antigens, such as the case of the HA rod antigen in influenza, thereby producing a more universal vaccine.

[0063] The use of a combination of TLR agonists as adjuvants generally results in an overall enhanced immune response, but in the case of vaccines for infectious diseases such as influenza, the Th1 (cell-mediated) immune response is enhanced or biased towards the Th1 type of immune response at the expense of the Th2 (humoral or antibody) type. In fact, despite the increase in Th1 response, this sometimes results in insufficient production of protective Th2 antibodies, and for influenza infection, a certain protective antibody titer is considered to be the main factor in providing effective protection through immunization.

[0064] In the present invention, it was found that the combined ratio of TLR4 / TLR7 agonists in a single nanoparticle formulation not only enhanced the overall immune response to the antigen, but also provided sufficient protective antibody production to effectively protect mice from the attack of the deadly virus. The immune state of humans will be very different from that of mice, who are usually new to antigens such as influenza, while humans are usually exposed to influenza antigens for many years through natural exposure and vaccine exposure. The same is true for other infectious pathogens such as chickenpox (varicella zoster), which may subsequently appear in humans in the form of shingles.

[0065] It is well known that immunization with one antigen blocks a strong immune response to a second, similar antigen. This may be due to 1) epitope rejection, in which pre-existing antibodies, especially mucosal IgA, protect the vaccine from all antigen-presenting cells; 2) reduced dendritic cell (DC) access, in which memory B cells internalize the new vaccine, reducing DC access and activation and T cell immunity; 3) T cell competition, in which memory B cells are activated, consume cytokines, cofactors and capture T cells that can react with antigen-loaded DC.

[0066] The present invention overcomes these deficiencies by: 1) encapsulating the vaccine in liposome nanoparticles to preferentially deliver the vaccine to DCs; 2) activating DCs using a specific ratio of combined TLR agonists, which will increase the number and diversity of activated T cells against vaccine antigens.

[0067] definition

[0068] When a composition contains at least about 90%, at least about 95%, 99%, and 99.9% (by weight) of a particular composition, then the composition is "substantially all" composed of a particular compound or a particular form of a compound (e.g., an isomer). When each compound (e.g., isomer) accounts for at least about 10% of the composition by weight, the composition contains a "mixture" of various compounds or various forms of the same compound. The TLR7 agonists or conjugates thereof of the present invention can be prepared as acidic or basic salts, as well as free acid or free base forms. In solution, certain compounds of the present invention may exist as zwitterions, in which the counterions are provided by the solvent molecules themselves, or by other ions dissolved or suspended in the solvent.

[0069] The term "toll-like receptor agonist" (TLR agonist) refers to a molecule that binds to a TLR. Synthetic TLR agonists are compounds designed to bind to a TLR and activate the receptor.

[0070] In the present invention, it should be understood that the compound of formula (I) or (II) or its salt may exhibit tautomerism, whereby the two compounds can be easily converted to each other by exchanging hydrogen atoms between the two atoms and forming a covalent bond with any of the two atoms. Since tautomeric compounds are in a mobile equilibrium state with each other, they can be regarded as different isomeric forms of the same compound. It should be understood that the structural formula graphics in this specification can only represent one of the possible tautomeric forms. However, it should also be understood that the present invention includes any tautomeric form and is not limited to any tautomeric form used in the structural formula graphics. The structural formula graphics in this specification can only represent one possible tautomeric form, and it should be understood that this specification covers all possible tautomeric forms of the drawn compound, not just those forms conveniently illustrated herein. For example, tautomerism may be manifested as a bonded pyrazolyl as shown by a wavy line. Although both substituents are referred to as 4-pyrazolyl, it is clear that different nitrogen atoms in each structure carry hydrogen atoms.

[0071]

[0072] Substituted pyrazoles such as 3-methyl, 5-methyl or 3,5-dimethylpyrazole also undergo this tautomerism. Another example of tautomerism is amide-imino (lactam-lactimide when cyclic) tautomerism, such as seen in heterocyclic compounds with ring oxygen atoms adjacent to ring nitrogen atoms. For example, the equilibrium:

[0073] is an example of tautomerism. Therefore, structures described herein as one tautomer also include the other tautomers.

[0074] Optical isomerism

[0075] It should be understood that when the compounds of the present invention contain one or more chiral centers, the compounds may exist and be separated in pure enantiomer or diastereoisomer form or as a racemic mixture. Therefore, the present invention includes any possible enantiomer, diastereomer, racemate or mixture thereof of the compounds of the present invention.

[0076] Isomers that arise due to the presence of chiral centers include a pair of non-superimposable isomers called "enantiomers". Single enantiomers of pure compounds are optically active, that is, they are able to rotate the plane of plane polarized light. Single enantiomers are named according to the Cahn-Ingold-Prelog system. The priority of the substituents is ranked according to atomic weight, with the higher the atomic weight determined by the system program, the higher the priority. Once the priority ranking of the four groups is determined, the molecule is oriented with the lowest ranked group away from the observer. The molecule is then named (R) if the descending order of the other groups proceeds clockwise and (S) if the descending order of the other groups proceeds counterclockwise. In the example of Scheme 14, the Cahn-Ingold-Prelog ranking is A>B>C>D. The lowest ranked atom D is oriented away from the observer.

[0077]

[0078] The present invention is intended to include diastereomers and their racemic and resolved forms, diastereomerically and enantiomerically pure forms and salts thereof.A pair of diastereomers may be resolved by known separation techniques, including normal and reverse phase chromatography and crystallization.

[0079] "Isolated optical isomers" refers to compounds that have been substantially purified from the corresponding optical isomers of the same structural formula. In one embodiment, the purity of the isolated isomers is at least about 80% by weight, such as at least 90%, 98% or 99%.

[0080] The separated optical isomers can be purified from the racemic mixture by well-known chiral separation techniques. According to one such method, a suitable chiral column, such as a chromatographic column, is used. A member of the family series (Daicel Chemical Industries Ltd., Tokyo, Japan), the racemic mixture of the compounds of the present invention or their chiral intermediates was separated by HPLC to give optical isomers with a purity of 99 wt%. The chromatographic column was operated according to the manufacturer's instructions.

[0081] The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are within the scope of reasonable medical judgment, suitable for use in contact with human and animal tissues, do not cause excessive toxicity, irritation, allergic reactions or other problems or complications, and have a reasonable benefit / risk ratio.

[0082] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds in which the parent compound is altered by preparing its acidic or basic salts. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid and the like; and salts prepared from organic acids such as 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.

[0083] Pharmaceutically acceptable salts useful for the compounds of the invention can be synthesized from parent compounds containing a basic or acidic unit by conventional chemical methods. Generally, these salts can be prepared by reacting the free acid or base of these compounds with a stoichiometric amount of a suitable base or acid in water or in an organic solvent or in a 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 at Remington' s Pharmaceutical Sciences ), 17th edition, Mack Publishing Company, Easton, Pennsylvania, p. 1418 (1985), the disclosure of which is incorporated herein by reference.

[0084] The compounds of the structural formula described herein can be solvates, and in some embodiments, can be hydrates. The term "solvate" refers to a solid compound having one or more solvent molecules combined with its solid structure. A solvate is formed when a compound is crystallized from a solvent. A solvate is formed when one or more solvent molecules become a component of a solid crystalline matrix during solidification. The compounds of the structural formula described herein can be solvates, such as ethanol solvates. Another solvate is a hydrate. "Hydrate" also refers to a solid compound having one or more water molecules that are closely combined with its solid or crystal structure at the molecular level. When a compound solidifies or crystallizes in water, a hydrate can be formed in which one or more water molecules become a component of the solid crystalline matrix.

[0085] Unless otherwise indicated, the following definitions apply: Halogen or halogen is fluorine, chlorine, bromine or iodine. Alkyl, alkoxy, alkenyl, alkynyl, etc. refer to straight and branched chain groups; however, reference to individual groups such as "propyl" includes only straight chain groups, and branched chain isomers such as "isopropyl" are specifically mentioned. Aryl refers to phenyl or an ortho-fused bicyclic carbocyclic group having about nine to ten ring atoms, at least one of which is aromatic. Het can be a heteroaryl group, which includes groups connected through the ring carbons of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and 1 to 4 heteroatoms, each heteroatom selected from non-peroxide oxygen, sulfur and N(X), wherein X is absent or is H, O, (C1-C4)alkyl, phenyl or benzyl, and groups of ortho-fused bicyclic heterocyclic rings of about eight to ten ring atoms derived therefrom, particularly benzyl derivatives or groups derived from fused propylene, trimethylene or tetramethylene diradicals.

[0086] It will be appreciated by those skilled in the art that the compounds of the invention having chiral centers can exist and be separated in optically active and racemic forms. Some compounds may exhibit allotropy. It will be appreciated that the invention includes any racemic, optically active, allotropic or stereoisomeric forms of the compounds of the invention having the useful properties described herein, or mixtures thereof, and how to prepare optically active forms (e.g., by resolving racemic forms by recrystallization techniques, synthesizing from optically active starting materials, chiral synthesis, or chromatographic separation using chiral stationary phases) and how to use the standards described herein, or other similar tests well known in the art to determine the activity of agonists are familiar to those skilled in the art. It will also be appreciated by those skilled in the art that the compounds described herein include their various tautomers, which can exist in various equilibrium states with each other.

[0087] The term "treat" as used herein refers to (i) preventing the occurrence of a pathological condition (e.g., prevention); (ii) inhibiting a pathological condition or preventing its development; (iii) alleviating a pathological condition; and / or (iv) improving, alleviating, mitigating, and eliminating symptoms of a disease. The amount of the candidate molecule or compound described herein in a formulation or medicament is an amount that can lead to a biological effect or lead to improvement, mitigation, alleviation, relief, attenuation, or elimination of symptoms of a condition (e.g., a disease). This term can also refer to reducing or preventing the rate of cell proliferation (e.g., slowing or preventing 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 microorganisms (bacteria) in a system (e.g., a cell, tissue, or subject) infected by a microorganism, reducing the rate of microbial reproduction, reducing the number of symptoms associated with a microbial infection or the impact of the symptoms, and / or removing the number of detectable microorganisms from a system. Examples of microorganisms include, but are not limited to, viruses, bacteria, and fungi.

[0088] The term "therapeutically effective dose" as used herein refers to an amount of a compound or a combination of compounds that treats or prevents a disease or disorder or treats a symptom of a disease or disorder in a subject. The terms "subject" and "patient" as used herein generally refer to an individual who will or has been treated with the methods described herein (e.g., administered a compound).

[0089] "Stable compound" and "stable structure" are meant to indicate 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 herein.

[0090] The term "subject," "patient," or "subject in need thereof" refers to a living organism suffering from or susceptible to a disease or condition that can be treated by administering a compound, pharmaceutical composition, mixture, or vaccine provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, cattle, 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 livestock 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 cattle. In some embodiments, the patient is a canine. In some embodiments, the patient is a feline. 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 an experimental 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 person. In some embodiments, the patient is an elderly mammal. In some embodiments, the patient is an elderly patient.

[0091] The term "effective dose" as used herein refers to an amount effective to achieve the intended purpose. Thus, the term "therapeutically effective dose" and the like as used herein refers to an amount of a compound, mixture or vaccine, or a combination thereof, that treats or prevents a disease or disorder, or treats a symptom of a disease or disorder, in a subject in need thereof.

[0092] The term "TLR" refers to Toll-like receptors, which are K B Components of the activated innate immune system.

[0093] The terms "TLR modulator", "TLR immunomodulator" and the like as used herein refer to compounds that agonize or antagonize 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 agonizes a TLR, while a "TLR antagonist" is a TLR modulator that antagonizes a TLR.

[0094] The term "TLR4" as used herein refers to the product of the TLR4 gene and its homologs, subtypes and functional fragments: subtype 1 (NCBI accession number NP_612564.1); subtype 2 (NCBI accession number NP_003257.1); subtype 3 (NCBI accession number NP_612567.1). TLR4 agonists that can be included in the disclosed formulations include, but are not limited to, compounds of formula (II), such as pyrimidoindoles, aminoalkylaminoglucosides phosphates, such as CRX-601 and CRX-547, RC-29, monophosphoryl lipid A (MPL), pyranosyl glucopyranose lipid adjuvants (GLA and SLA), OM-174, PET lipid A, ONO-4007, INI-2004 (diamine allose phosphate) and E6020.

[0095] The term "TLR7" as used herein refers to the product of the TLR7 gene and its homologues and functional fragments (NCBI Accession No. AAZ99026). 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 gardiquimid, CL264, adenine analogs such as CL087, thiazoquinolines such as 3M002 (CL075), guanosine analogs such as loxoribine, or thioquinolines.

[0096] TLR4 and TLR7

[0097] Toll-like receptors (TLRs) are pattern recognition receptors that recognize conserved microbial products, called pathogen-associated molecular patterns (PAMPs). TLR4 recognizes LPS. TLR4 signaling activates MyD88- and TRIF-dependent pathways. The MyD88 pathway activates NFNF-κB and JNK to induce inflammatory responses. The TRIF pathway activates IRF3 to induce IFN-α production.

[0098] TLR4 is mainly expressed on monocytes, mature macrophages and dendritic cells, mast cells and intestinal epithelial cells. TLR4 TLR modulators (antagonists) 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., al., Mol. Pharmacol. 2006, 69: 1288-1295). TLR4 TLR modulators (agonists) include 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 the MyD88-dependent pathway and the IRF7-dependent signaling. The IRF7 pathway induces IFN-α production.

[0099] TLR7 senses ss-RNA or synthetic chemicals (imiquimod, R848). TLR7 and TLR8 are present in the endosomes of monocytes and macrophages, TLR7 is also expressed on plasmacytoid dendritic cells, and TLR8 is also expressed in mast cells. Both receptors can recognize single-stranded RNA of viruses. Synthetic ligands, such as R-848 and imiquimod, can be used to activate TLR7 and TLR8 signaling pathways. 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 in bacterial and viral DNA. Synthetic oligonucleotides comprising 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 induce very little interferon-a (IFNa). Class C oligonucleotides target plasmacytoid dendritic cells and are effective inducers of IFNa. This type of oligonucleotide participates in the activation and maturation of antigen presenting cells, indirectly activates natural killer cells and directly stimulates B cells. See, for example, Vollmer, J., et al., Eur. J. Immunol. 2004, 34: 251-262; Strandskog, G., et al., Dev. Comp. Immunol. 2007, 31: 39-51.

[0100] Reported TLR7 modulators (agonists) 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.). TLR8 TLR modulators (agonists) include VTX-1463 (Hennessey 2010, Id.). TLR7 and TLR8 TLR modulators (agonists) 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) of 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), 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).

[0101] TLR7 ligands and conjugates thereof

[0102] When referring to TLR7 ligands and conjugates thereof, the terms "alkyl", "alkenyl" and "alkynyl" described herein may include linear, branched and cyclic monovalent hydrocarbon groups, and combinations thereof, which contain only C and H when unsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, etc. The total number of carbon atoms in each such group is sometimes described herein, for example, when the group may contain up to ten carbon atoms, it may be 1-10C or C1-C 10 or C 1-10 For example, when heteroatoms (usually N, O and S) are allowed to replace carbon atoms in a heteroalkyl group, the number describing the group, while still written as, for example, C1-C6, represents the total number of carbon atoms in the group plus the number of said heteroatoms included as replacements for carbon atoms in the backbone of the ring or chain being described.

[0103] 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). For example, they contain one 4C(alkyl) or two 4C(alkenyl or alkynyl). A monoradical may include more than one multiple bond, or more than one multiple bond; when they contain at least one carbon-carbon double bond, these groups are included in the definition of the term "alkenyl", and when they contain at least one carbon-carbon triple bond, these groups are included in the term "alkynyl".

[0104] Alkyl, alkenyl and alkynyl groups are typically optionally substituted to the extent that the substitution makes chemical sense. Typical substituents include, but are not limited to, halogen, =O, =N-CN, =N-OR, =NR, OR, NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, OOCR, COR and NO2, wherein each R is independently 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 10 Aryl or C5-C 10 heteroaryl, and R is optionally substituted with halogen, =O, =N-CN, =N-OR', =NR', OR', NR'2, SR', SO2R', SO2NR'2, NR'SO2R', NR'CONR'2, NR'COOR', NR'COR', CN, COOR', CONR'2, OOCR', COR' and NO2, wherein R' is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl or C5-C10 Heteroaryl. Alkyl, alkenyl and alkynyl may also be replaced by C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl or C5-C 10 The heteroaryl group is substituted, wherein each substituent may be substituted with substituents that are appropriate for that particular group.

[0105] "Alkynyl" substituents may include optionally substituted 2-10C alkynyl and have 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 Arylalkyl or C6-C 12 wherein each R' is independently H, C1-C6 alkyl, C2-C6 heteroalkyl, C1-C6 acyl, C2-C6 heteroacyl, C6-C6 heteroalkyl, C1-C6 acyl, C2-C6 heteroalkyl ... 10 Aryl, C5-C 10 Heteroaryl, C 7-12 Arylalkyl or C 6-12 Heteroaralkyl, wherein each of them is optionally substituted by one or more groups selected from halogen, C1-C4 alkyl, C1-C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl, hydroxyl, amino and =O; and wherein two R's may be linked to form a 3-7 membered ring optionally containing no more than 3 heteroatoms selected from N, O and S. In one embodiment, Ri in -C≡C-Ri is H or Me.

[0106] "Heteroalkyl", "heteroalkenyl" and "heteroalkynyl" are defined similarly to the corresponding hydrocarbyl groups (alkyl, alkenyl and alkynyl), but the "hetero" term refers to a group containing 1 to 3 O, S or N heteroatoms or combinations thereof within the backbone residue; thus, at least one carbon atom of the corresponding alkyl, alkenyl or alkynyl group is replaced with one of the specified heteroatoms to form a heteroalkyl, heteroalkenyl or heteroalkynyl group. The typical sizes of heteroforms of alkyl, alkenyl and alkynyl groups are generally the same as those of the corresponding hydrocarbyl groups, and the substituents that may be present on the heteroforms are the same as those described above for the hydrocarbyl groups. For reasons of chemical stability, it is also understood that such groups do not include more than two consecutive heteroatoms unless otherwise specified, unless an oxo group is present on N or S, such as in a nitro or sulfonyl group.

[0107] Although "alkyl" as used herein includes cycloalkyl and cycloalkylalkyl, the term "cycloalkyl" may be used herein to describe a carbocyclic non-aromatic group connected through a ring carbon atom, and "cycloalkylalkyl" may be used to describe a carbocyclic non-aromatic group connected to a molecule through an alkyl linker. Similarly, "heterocyclyl" may be used to describe a non-aromatic ring group containing at least one heteroatom as a ring member and connected to a molecule through a ring atom, which may be C or N; and "heterocyclylalkyl" may be used to describe such a group connected to another molecule through a linker. The size and substituents applicable to cycloalkyl, cycloalkylalkyl, heterocyclyl and heterocyclylalkyl are the same as those described above for alkyl. These terms as described herein also include rings containing one or two double bonds, as long as the ring is not aromatic.

[0108] As used herein, "acyl" includes groups comprising an alkyl, alkenyl, alkynyl, aryl or aralkyl group attached at one of the two available valence positions of the carbonyl carbon atom, and heteroacyl refers to the corresponding group in which at least one carbon other than the carbonyl carbon has been replaced by a heteroatom selected from N, O and S. Thus heteroacyl includes, for example, -C(=O)OR and -C(=O)NR2 as well as -C(=O)-heteroaryl.

[0109] Acyl and heteroacyl are connected to any group or molecule to which they are connected through the open valence of the carbonyl carbon atom. Typically, they are C1-C8 acyl, including formyl, acetyl, pivaloyl and benzoyl, and C2-C8 heteroacyl, including methoxyacetyl, ethoxycarbonyl and 4-pyridoyl. Hydrocarbyl, aryl and heteromorphisms of such groups comprising acyl or heteroacyl can be substituted with substituents described herein that are typically suitable substituents for each corresponding portion of acyl or heteroacyl.

[0110] "Aromatic" or "aryl" moieties refer to monocyclic or fused bicyclic moieties having well-known aromatic characteristics; examples include phenyl and naphthyl. Similarly, "heteroaromatic" and "heteroaryl" refer to monocyclic or fused bicyclic ring systems containing one or more heteroatoms selected from O, S and N as ring members. The inclusion of heteroatoms allows aromaticity in 5-membered rings as well as 6-membered rings. Typical heteroaromatic systems include monocyclic C5-C6 aryl groups such as pyridyl, pyrimidinyl, pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl and imidazolyl, as well as C8-C6 aryl groups formed by fusing one of these monocyclic groups to a benzene ring or to any heteroaromatic monocyclic group. 10 The fused bicyclic portion of a bicyclic group, for example, indolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, pyrazolopyridinyl, quinazolinyl, quinoxalinyl, cinnolinyl, etc. Any monocyclic or fused bicyclic ring system having aromatic characteristics in terms of the electron distribution of the entire ring system is included in this definition. It also includes bicyclic groups in which at least the ring directly connected to the rest of the molecule has aromatic characteristics. Typically, the ring system contains 5-12 ring member atoms. For example, a monocyclic heteroaryl may contain 5-6 ring members, while a bicyclic heteroaryl may contain 8-10 ring members.

[0111] The aryl and heteroaryl moieties may be substituted with a variety of substituents, including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C 12 Aryl, C1-C8 acyl and hetero forms of these substituents, each of which can itself be further substituted; other substituents for aryl and heteroaryl moieties include halogen, OR, NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, OOCR, COR and NO2, wherein each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C7-C 12 Arylalkyl or C6-C 12 alkyl, and each R is optionally substituted as described above for alkyl. Substituents on aryl or heteroaryl can of course be further substituted with groups described herein as suitable for each type of such substituent or each portion of such substituent. Thus, for example, an aralkyl substituent can be substituted on the aryl portion with typical substituents for aryl described herein, and it can be further substituted on the alkyl portion with typical or suitable substituents for alkyl described herein.

[0112] Similarly, "aralkyl" and "heteroaralkyl" refer to aromatic and heteroaromatic ring systems bonded to their points of attachment by a linking group, such as an alkylene group, including substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic linking groups. Typically the linking group is a C1-C8 alkyl group or its heteromorphism. These linking groups may also include a carbonyl group, thereby enabling them to provide substituents as acyl or heteroacyl moieties. The aryl or heteroaryl rings in the aralkyl or heteroaralkyl groups may be substituted with the same substituents described for the aryl groups. For example, an aralkyl group includes a benzene ring, optionally substituted with the groups defined above for the aryl groups and substituted with unsubstituted or C1-C4 alkylene groups substituted with one or two C1-C4 alkyl or heteroalkyl groups, wherein the alkyl or heteroalkyl groups may optionally be cyclized to form a ring, such as cyclopropane, dioxolane or oxacyclopentane. Similarly, heteroaralkyl may include a C5-C6 monocyclic heteroaryl group optionally substituted by the above-mentioned typical substituents for aryl and a C1-C4 alkylene group which is unsubstituted or substituted by one or two C1-C4 alkyl or heteroalkyl groups, or include an optionally substituted benzene ring or a C5-C6 monocyclic heteroaryl group and a C1-C4 heteroalkylene group which is unsubstituted or substituted by one or two C1-C4 alkyl or heteroalkyl groups, wherein the alkyl or heteroalkyl group may be optionally cyclized to form a ring, for example, cyclopropane, dioxolane or oxacyclopentane.

[0113] Where aralkyl or heteroaralkyl is described as optionally substituted, the substituents may be on the alkyl or heteroalkyl portion or the aryl or heteroaryl portion of the group. The substituents optionally present on the alkyl or heteroalkyl portion are generally the same as those described above for alkyl; the substituents optionally present on the aryl or heteroaryl portion are generally the same as those described above for aryl.

[0114] "Aralkyl" groups as described herein are hydrocarbon groups if they are unsubstituted and are described by the total number of carbon atoms in the ring and alkylene or similar linking group. Thus, benzyl is a C7-aralkyl, while phenethyl is a C8-aralkyl.

[0115] As described above, "heteroaralkyl" refers to a moiety containing an aryl group connected by a linking group, and is different from "aralkyl" in that at least one ring atom of the aryl moiety or one atom in the linking group is a heteroatom selected from N, O and S. Heteroaralkyls are described herein according to the total number of atoms in the ring and linking group combined, and they include aryl groups connected by a heteroalkyl linking group; heteroaryl groups connected by a hydrocarbyl linking group such as an alkylene group; and heteroaryl groups connected by a heteroalkyl linking group. Thus, for example, C7-heteroaralkyl would include pyridylmethyl, phenoxy, and N-pyrrolylmethoxy.

[0116] As used herein, "alkylene" refers to a divalent hydrocarbon group; because it is divalent, it can link two other groups together. Usually it refers to -(CH2) n-, where n is 1-8, for example n is 1-4, although where specified, alkylene may also be substituted with other groups and 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- may also be referred to as alkylene, as may cyclic groups such as cyclopropane-1,1-diyl. When alkylene is substituted, the substituents include those normally present on the alkyl groups described herein.

[0117] In general, any alkyl, alkenyl, alkynyl, acyl or aryl or aralkyl group contained in a substituent or any heteromorph of one of these groups may itself be optionally substituted with further substituents. If no substituents are otherwise described, the properties of these substituents are similar to those listed for the main substituents themselves. Thus, in, for example, R 2 In the embodiment where R is an alkyl group, the alkyl group may optionally be 2 The remaining substituents listed in the examples are substituted, where such should make chemical sense and not violate the size restrictions for the alkyl group itself; for example, an alkyl group substituted by an alkyl group or by an alkenyl group would simply extend the carbon atom upper limit of these examples and is not included. However, alkyl groups substituted by aryl groups, amino groups, alkoxy groups, =O groups, etc. are also included within the scope of the present invention, and the number of atoms of these substituents is not included in the number of atoms used to describe the alkyl, alkenyl, etc. groups. In the absence of a specified number of substituents, each such alkyl, alkenyl, alkynyl, acyl or aryl group can be substituted with as many substituents as are available for it; in particular, for example, any of these groups can be substituted with fluorine atoms at any or all of its available valences.

[0118] In various embodiments, the present invention provides a method for preventing, inhibiting or treating liver diseases, such as liver diseases associated with inflammation in mammals. The method comprises administering to a mammal in need thereof an effective dose of a compound of formula (I):

[0119]

[0120] Where X 1 Is -O-, -S- or -NR c -;

[0121] 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 Heterocyclic group;

[0122] R c It is hydrogen, C 1-10 Alkyl, or substituted C1-10 Alkyl; or R c and R 1 Together with the nitrogen to which they are attached, they form a heterocyclic ring or a substituted heterocyclic ring;

[0123] 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)-(C1-C6) 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), halogen, nitro or cyano, or no R 2 ;

[0124] 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;

[0125] Where any substituent on the alkyl, aryl or heterocyclic group is hydroxyl, C 1-6 Alkyl, hydroxyl C 1-6 Alkylene, C 1-6 Alkoxy, C 3-6 Cycloalkyl, C 1-6 Alkoxy C 1-6 Alkylene, amino, cyano, halogen or aryl;

[0126] n is 0, 1, 2, 3 or 4.

[0127] X 2 is a bond or a linking group; and

[0128] In one embodiment, R x Contains one or two carboxylates, or contains –(R 3 ) r –(R 4 ) s )p of phospholipids, each of which R 3 is independently a polyethylene glycol (PEG) moiety; wherein each 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 , 5-6 membered ring, substituted 5-6 membered ring, -C1-C6 alkyl-5-6 membered ring, -C1-C6 alkyl-substituted 5-6 membered ring C2-C9 heterocyclyl, or substituted C2-C9 heterocyclyl; wherein r is 1 to 1000, wherein s is 1 to 100, and wherein p is 1 to 100;

[0129] or their tautomers;

[0130] or a pharmaceutically acceptable salt or solvate thereof.

[0131] In one embodiment, R 3 is the PEG moiety.

[0132] In some embodiments, the PEG reactant has the structure CH3O(CH2CH2O) n -X-NHS*, where X can be -COCH2CH2COO-, -COCH2CH2CH2COO-, -CH2COO-, and –(CH2)5COO-. In certain embodiments, the PEG reactant has the structure:

[0133]

[0134] In some embodiments, certain PEG reactants are bifunctional. In some embodiments, examples of bifunctional PEG reactants have the structure X-(OCH2CH2)n-X, where X is (N-succinimidyloxycarbonyl)methyl (-CH2COO-NHS), succinimidylglutarate (-COCH2CH2CH2COO-NHS), (N-succinimidyloxycarbonyl)pentyl (-(CH2)5COO-NHS), 3-(N-maleimido)propionamido, (-NHCOCH2CH2-MAL), aminopropyl (-CH2CH2CH2NH2), or 2-mercaptoethyl (-CH2CH2SH).

[0135] In certain embodiments, some PEG reactants are heterofunctional. Examples of heterofunctional PEG reactants have the structure

[0136]

[0137] In some embodiments, X can be (NN-succinimidyloxycarbonyl)methyl (-CH2COO-NHS), succinimidylglutarate (-COCH2CH2CH2COO-NHS), (N-succinimidyloxycarbonyl)pentyl (-(CH2)5COO-NHS), 3-(N-maleimido)propionamide, (-NHCOCH2CH2-MAL), 3-aminopropyl (-CH2CH2CH2NH2), 2-mercaptoethyl (-CH2CH2SH), 5-(N-succinimidyloxycarbonyl)pentyl (-(CH2)5COO-NHS], or p-nitrophenoxycarbonyl, (-CO2-p-C6H4NO2).

[0138] Certain branched chain PEG reactants may also be used, such as those having the following structure:

[0139]

[0140] In some embodiments, wherein X is a spacer group and Y is a functional group, including but not limited to maleimide, amine, glutaryl-NHS, carbonate-NHS or carbonate-p-nitrophenol. One advantage of branched chain PEG reactants is that they can produce conjugated products with sustained release properties.

[0141] In some embodiments, the PEG reactant may also be a heterofunctional reactant, such as

[0142] HO(CH2CH2O) n -CH2CH2CH2NH2,

[0143] HCl·H2N-CH2CH2CH2O(CH2CH2O) n -(CH2)5COOH and

[0144] HO(CH2CH2O) n -CH2CH2CHO.

[0145] In some embodiments, Boc * -Protected-amino-PEG-carboxy-NHS or maleimide-PEG-carboxy-NHS reactants.

[0146] In certain embodiments, comb polymers can be used as PEG reactants to incorporate multiple PEG units into the conjugate. Examples of comb polymers are shown below.

[0147]

[0148] In some embodiments, the PEG reactants and / or PEG conjugate products may have a molecular weight ranging from about 5 g / mol to about 100,000 g / mol. In some embodiments, the average, median or nominal molecular weight of the PEG reactants and / or PEG conjugate products is 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 g / mol. In some embodiments, the PEG moiety in the compounds herein is homogeneous, and the molecular weight of the PEG moiety is the same for each molecule of a particular batch of compounds (e.g., R 3 is a PEG unit, r is 2 to 10).

[0149] In various embodiments, X in formula (I) 2 It can be a chain having from 1 to about 10 atoms in a bond or chain, wherein the atoms of the chain are selected from carbon, nitrogen, sulfur and oxygen, wherein any carbon atom may be substituted by oxygen, wherein any sulfur atom may be substituted by one or two oxy groups. The chain may be interrupted by one or more cycloalkyl, aryl, heterocyclyl or heteroaryl rings.

[0150] In formula (I), X 2 Some non-limiting examples include – (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 -, wherein each y (subscript) and z (superscript) is independently 0 to 20, and each Y and Z is independently 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 10Aryl, C5-C9 heterocyclic group, substituted C5-C9 heterocyclic group, C1-C6 alkanoyl, Het, HetC1-C6 alkyl or C1-C6 alkoxycarbonyl, wherein the substituent on the alkyl, cycloalkyl, alkanoyl, alkoxycarbonyl, Het, aryl or heterocyclic group is hydroxyl, C1-C 10 Alkyl, hydroxyl C1-C 10 Alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9 heterocyclic, C 1-6 Alkoxy C 1-6 In some embodiments, the linker is sometimes a -C(Y')(Z')-C(Y")(Z")-linker, wherein each Y', Y", Z' and Z" is independently 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 group, substituted C5-C9 heterocyclic group, C1-C6 alkanoyl, Het, HetC1-C6 alkyl or C1-C6 alkoxycarbonyl, wherein the substituent on the alkyl, cycloalkyl, alkanoyl, alkoxycarbonyl, Het, aryl or heterocyclic group is hydroxyl, C1-C 10 Alkyl, hydroxyl C1-C 10 Alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9 heterocyclic, C 1-6 Alkoxy C 1-6 alkenyl, amino, cyano, halogen or aryl.

[0151] In formula (I), X 2 Another special value is

[0152]

[0153]

[0154] X 2 Another special value is

[0155]

[0156] In various embodiments, X 2 It can be C(O), or it can be

[0157]

[0158] Any one of .

[0159] In various embodiments, X in Formula (I) 1 It can be oxygen.

[0160] In various embodiments, X in formula (I) 1 It can be sulfur, or it can be -NR c -, where R c It is hydrogen, C 1-6 Alkyl or substituted C 1-6 Alkyl, wherein the alkyl substituent is hydroxyl, C 3-6 Cycloalkyl, C 1-6 More specifically, X 1 It can be -NH-.

[0161] In various embodiments, R in formula (I) 1 and R c Together they may form a heterocyclic or substituted heterocyclic ring. More specifically, R 1 and R c Together they may form a substituted or unsubstituted morpholine, piperidine, pyrrolidine or piperazine ring.

[0162] In various embodiments, R in formula (I) 1 Can be C 1-6 Alkoxy substituted C1-C 10 alkyl.

[0163] In various embodiments, R in formula (I) 1 It can be hydrogen, C 1-4 Alkyl or substituted C 1-4 More specifically, R 1 It can be hydrogen, methyl, ethyl, propyl, butyl, hydroxyl C 1-4 Alkylene or C 1-4 Alkoxy C 1-4 More specifically, R 1 It may be hydrogen, methyl, ethyl, methoxyethyl or ethoxyethyl.

[0164] In various embodiments, R in formula (I) 2 Can be absent, or R 2 Can be halogen or C 1-4 More specifically, R 2 It can be chlorine, bromine, methyl or ethyl.

[0165] In one embodiment, R x Yes((R 3 ) r –(R 4 ) s ) p or R3 In one embodiment, R 3 is a PEG moiety or a PEG moiety derivative. In certain embodiments, R 3 In some embodiments, the PEG moiety may include one or more PEG units. In some embodiments, the PEG moiety may include about 1 to about 1000 PEG units, 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 PEG units. In certain embodiments, the PEG moiety can include about 1 to 5 to about 25 PEG units, about 1 to 5 to about 10 PEG units, about 10 to about 50 PEG units, about 18 to about 50 PEG units, about 47 to about 150 PEG units, about 114 to about 350 PEG units, about 271 to about 550 PEG units, about 472 to about 950 PEG units, about 50 to about 150 PEG units, about 120 to about 350 PEG units, about 250 to about 550 PEG units, or about 650 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 5-6 membered ring, a substituted 5-6 membered ring, a -C1-C6 alkyl-5-6 membered ring, a -C1-C6 alkyl-substituted 5-6 membered ring, a C2-C9 heterocycle, or a substituted C2-C9 heterocycle.

[0166] In some embodiments, r is from about 5 to about 100, 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, sometimes r is about 10. In some embodiments, R 3 It is the PEG unit (PEG) r , and r is from about 2 to about 10 (e.g., r is from about 2 to about 4) or from about 18 to about 500.

[0167] In some embodiments, s is from about 5 to about 100, 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, 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. For a linear portion, s is sometimes less than r (e.g., when R3 is -O-CH2-CH2- or -CH2-CH2-O-), and sometimes s is 1. In some embodiments, R 3 is a linear PEG moiety (e.g., having about 1 to about 1000 PEG units), s is 1 and r is 1. For branched moieties, s is sometimes less than, greater than, or equal to r (e.g., when R 3 is -O-CH2-CH2- or -CH2-CH2-O-), 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).

[0168] In some embodiments, R 3 is -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).

[0169] In certain embodiments, X 2 is an acylamino linking group (e.g., -C(O)NH- or -NH(O)C-); an alkylacylamino 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-C(O)NH-C1-C6 alkyl- or -C(O)NH-(CH2) t-, wherein t is 1, 2, 3 or 4); a substituted 5-6 membered ring (e.g., an aromatic ring, a heteroaromatic ring (e.g., a tetrazole, a pyridyl, a 2,5-pyrrolidinedione (e.g., a 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).

[0170] As used herein, the term "phospholipid" refers to a mono- or di-glyceride with a phosphate group bonded to a glycerol hydroxyl group, wherein an alkanolamine group is bonded as an ester to the phosphate group, having the following general formula:

[0171]

[0172] Where R 11 and R 12 are each independently hydrogen or acyl, and depending on pH, R 13 is a negative charge or hydrogen. When R 13 When is negatively charged, a suitable counterion may be present, such as a sodium ion. For example, the alkanolamine moiety may be an ethanolamine moiety, such that m=1. It is also understood that the NH group may be protonated and positively charged, or unprotonated and neutral, depending on the pH. For example, a phospholipid may exist as a zwitterion having a negatively charged phosphate oxygen anion and a positively charged protonated nitrogen atom. 12 The carbon atom of is a chiral carbon atom, so the molecule can exist as an R isomer, an S isomer, or any mixture thereof. When equal amounts of R and S isomers are present in a sample of the compound of formula (II), the sample is referred to as a "racemate". For example, in the commercially available product 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, R 3 The group is chiral

[0173] This is the R absolute configuration.

[0174] Phospholipids can be free molecules or covalently linked to another group, such as shown in the figure

[0175] The wavy lines represent bonding points.

[0176] Thus, when a substituent, such as R x When referred to as a phospholipid, this means that the phospholipid group is bonded to another group, such as an N-benzyl heterocyclic ring system disclosed herein, as specified by the structure. Unless otherwise indicated, such as in the structural description, the point of attachment of the phospholipid group can be at any chemically feasible position. For example, in the phospholipid structure shown above, the point of attachment to another chemical moiety can be through the ethanolamine nitrogen atom, such as through an amide group bonded to the carbonyl group of another chemical moiety, such as

[0177] Where R is another chemical moiety that is bound to the phospholipid. In this bonded amide derivative, R 13 The group may be a proton or may be negatively charged and linked to a counterion such as a sodium ion. The acylated nitrogen atom of the alkanolamine group is no longer a basic amine but a neutral amide and therefore will not be protonated at physiological pH.

[0178] As used herein, the term "acyl" refers to an organic structure with a carbonyl group that is bonded to, for example, a glycerol hydroxyl group of a phospholipid via the carbonyl group to form a "carboxylate" group. Examples of acyl groups include fatty acid groups, such as oleoyl, which thus form a fatty (e.g., oleoyl) ester with a glycerol hydroxyl group. Thus, when R 11 or R 12 , but not both are acyl groups. The phospholipids shown above are monocarboxylates. 11 and R 12 When all are acyl groups, the phospholipids shown above are dicarboxylates.

[0179] In one embodiment, R x The phospholipids contain two carboxylates, each carboxylate including 1, 2, 3 or 4 sites of unsaturation, epoxidation, hydroxylation or a combination thereof.

[0180] In one embodiment, R x The phospholipids contain two carboxylates, which may be the same or different.

[0181] In one embodiment, each carboxylate of the phospholipid is a C17 carboxylate having the site of unsaturation at C8-C9.

[0182] In one embodiment, each carboxylate of the phospholipid is a C18 carboxylate having a site of unsaturation at C9-C10.

[0183] In one embodiment, X 2 It may be a bond or a chain having from 1 to about 10 atoms in the chain, wherein the atoms of the chain are selected from carbon, nitrogen, sulfur and oxygen, wherein any carbon atom may be substituted by oxygen, and wherein any sulfur atom may be substituted by one or two oxygen atoms.

[0184] In one embodiment, X 2 is C(O),

[0185]

[0186] In one embodiment, R x Includes dioleoylphosphatidylethanolamine (DOPE).

[0187] In one embodiment, R xis 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, X 2 It is C(O).

[0188] In one embodiment, X 1 It is oxygen or -NH-.

[0189] In one embodiment, R 1 and R c Together they form a heterocyclic or substituted heterocyclic ring, for example, a substituted or unsubstituted morpholine, piperidine, pyrrolidine or piperazine ring.

[0190] In one embodiment, R 1 Is C 1-6 Alkoxy-substituted C1-C10 alkyl, R 1 It is hydrogen, C 1-4 Alkyl or substituted C 1-4 Alkyl, R 1 is hydrogen, methyl, ethyl, propyl, butyl, hydroxyl C 1-4 Alkylene or C 1-4 Alkoxy C 1-4 Alkylene, or R 1 It is hydrogen, methyl, ethyl, methoxyethyl or ethoxyethyl.

[0191] In one embodiment, the composition further comprises an amount of an antigen.

[0192] In various embodiments, the mammal can be a human.

[0193] In various embodiments, the composition may be administered intranasally, or may be administered transdermally, or may be administered systemically.

[0194] TLR4 ligands

[0195] As used with respect to TLR4 ligands, the term "alkyl", by itself or as part of another substituent, unless otherwise indicated, refers to a straight chain (i.e., unbranched) or branched chain, or combinations thereof, which may be fully saturated, monounsaturated, or polyunsaturated and may include divalent and polyvalent groups, having a specified number of carbon atoms (i.e., C1-C 10The term "alkyl" refers to 1 to 10 carbons. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, (cyclohexyl) methyl, their homologues and isomers, such as, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl radicals are alkyl radicals with one or more double or triple bonds. Examples of unsaturated alkyl radicals include, but are not limited to, vinyl, 2-propenyl, butenyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl and higher homologues and isomers. Alkoxy is an alkyl radical that is connected to the rest of the molecule by an oxygen linking group (-O-).

[0196] Unless otherwise indicated, the term "alkylene" by itself or as part of another substituent refers to a divalent group derived from an alkyl group, such as, but not limited to, -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have 1 to 24 carbon atoms. In one embodiment, these groups have 10 or fewer carbon atoms. "Lower alkyl" or "lower alkylene" is an alkyl or alkylene group with a shorter chain, typically having eight or fewer carbon atoms.

[0197] Unless otherwise stated, the term "heteroalkyl" by itself or in combination with another term refers to a stable straight or branched chain consisting of at least one carbon atom and at least one heteroatom selected from O, N, P, Si and S, or combinations thereof, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom O, N, P, S and Si may be located at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the rest of the molecule. Examples include, but are not limited to:

[0198] -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.

[0199] Unless otherwise indicated, the term "heteroalkylene" by itself or as part of another substituent refers to a divalent radical derived from a heteroalkyl group, such as, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene, heteroatoms may also occupy one or both chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Further, 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)2R'- represents -C(O)2R'- and -R'C(O)2-. As noted above, heteroalkyl groups described herein include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R", -OR', -SR', and / or -S02R'. When referring to "heteroalkyl" followed by a listing of specific heteroalkyl groups, such as -NR'R", etc., it should be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the listing of specific heteroalkyl groups is for clarity. Thus, the term "heteroalkyl" herein should not be interpreted as excluding specific heteroalkyl groups, such as -NR'R", etc.

[0200] Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms refer to cyclic forms of "alkyl" and "heteroalkyl", respectively. In addition, for heterocycloalkyl, heteroatoms may occupy the position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidyl, 2-piperidyl, 3-piperidyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, 1-piperazinyl, 2-piperazinyl, etc. "Cycloalkylene" and "heterocycloalkylene" by themselves or as part of another substituent refer to divalent groups derived from cycloalkyl and heterocycloalkyl, respectively.

[0201] Unless otherwise indicated, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine or iodine atom. In addition, terms such as "haloalkyl" are intended 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, etc.

[0202] Unless otherwise indicated, the term "acyl" refers to -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.

[0203] Unless otherwise indicated, the term "aryl" refers to a polyunsaturated aromatic hydrocarbon substituent, which can be a monocyclic or fused together (i.e., fused ring aromatic) or covalently linked polycyclic (e.g., 1 to 3 rings). A fused ring aromatic refers to multiple rings fused together, wherein at least one fused ring is an aromatic ring. The term "heteroaryl" refers to an aromatic group (or ring) comprising at least one heteroatom selected from N, O, and S, wherein the nitrogen atom and the sulfur atom are optionally oxidized, and the nitrogen atom is optionally quaternized. Therefore, the term "heteroaryl" includes fused ring heteroaryls (i.e., multiple rings fused together, wherein at least one fused ring is a heteroaromatic ring). 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, wherein at least one ring is a heteroaromatic ring. Similarly, 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, wherein at least one ring is a heteroaromatic ring. In addition, 6,5-fused ring heteroarylene refers to two rings fused together, one of which has 6 members and the other has 5 members, at least one of which is a heteroaromatic ring. The heteroaryl group can be attached to the rest of the molecule through a carbon atom or a 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, 2-thiazolyl, 4 1- 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

[0204] For simplicity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, aralkyl) includes aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" is intended to include those groups in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), including those in which a carbon atom (e.g., a methylene group) has been replaced, for example, with an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.).

[0205] The term "oxo" as used herein refers to an oxygen double-bonded to a carbon atom.

[0206] The term "alkylsulfonyl" as used herein refers to a moiety having the structural formula -S(O2)-R', wherein R' is an alkyl group as defined above. R' may have a specified number of carbons (eg, "C1-C4 alkylsulfonyl").

[0207] Each of the above terms (eg, "alkyl," "heteroalkyl," "aryl," and "heteroaryl") include both substituted and unsubstituted forms of the designated radical.

[0208] Substituents for alkyl and heteroalkyl groups (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) may be one or more selected from, but not limited to, the following groups: -OR', =0, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -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, and their number is 0 to (2m'+1), where m' is the total number of carbon atoms in the group. In one embodiment, R', R", R"' and R"" each independently refers to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or aralkyl. When a compound of the invention includes more than one R group, for example, each R group is independently selected, just as each R', R", R"' and R"" group is independently selected when there is more than one R', R", R"' and R"" group. When R' and R" are attached to the same nitrogen atom, they can be combined 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. Based on the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is intended to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl groups (e.g., -CF3 and -CH2CF3) and acyl groups (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0209] Similar to the alkyl substituents described, aryl and heteroaryl substituents are varied and are selected from, for example: -OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -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, -NO2, -R', -N3, -CH(Ph)z, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, from 0 to the total number of open valences on the aromatic ring system; wherein 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, for example, each R group is independently selected, just as each R', R", R"', and R"" group is independently selected when there are more than one R', R", R"', and R"" groups.

[0210] Two or more substituents may be optionally connected to form aryl, heteroaryl, cycloalkyl or heterocycloalkyl. This so-called ring-forming substituent is usually (although not necessarily) connected to the cyclic base structure. In one embodiment, the ring-forming substituent is connected to the adjacent members of the base structure. For example, two ring-forming substituents connected to the adjacent members of the cyclic base structure form a condensed ring structure. In another embodiment, the ring-forming substituent is connected to a single member of the base structure. For example, two ring-forming substituents connected to a single member of the cyclic base structure form a spiro ring structure. In another embodiment, the ring-forming substituent is connected to the non-adjacent members of the base structure.

[0211] Two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally form the formula -TC(O)-(CRR') q -U-, wherein T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer from 0 to 3. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced by a substituent of the formula -A-(CH2) r-B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by a substituent of the formula -(CRR') s -X'-(C"R"') d -substituted with a substituent, wherein s and d can independently be 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.

[0212] As used herein, the term "heteroatom" or "ring heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and silicon (Si).

[0213] The term "substituent" as used herein refers to a group selected from the following:

[0214] (A) -OH, -NH2, -SH, -CN, -CF3, -CCl3, -NO2, oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0215] (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl substituted with at least one substituent selected from the group consisting of: (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 substituted with at least one substituent selected from the group consisting of: (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 and heteroaryl substituted with at least one substituent selected from the group consisting of: (a) oxo, -OH, -NH2, -SH, -CN, -CF3, -CCl3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl N, -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 substituted with at least one substituent selected from the group consisting of oxo, -OH, -NH2, -SH, -CN, -CF3, -CCl3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl and unsubstituted heteroaryl.

[0216] The "substituent group with limited size" or "substituent group with limited size" as used herein refers to a group selected from all substituent groups described in the "substituent group" above, wherein each substituted or unsubstituted alkyl group is a substituted or unsubstituted C1-C 20 Alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C4-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4-8 membered heterocycloalkyl.

[0217] The "lower substituent" or "lower substituent group" described herein refers to a group selected from all substituents described above for the "substituent group", wherein each substituted or unsubstituted alkyl is, for example, a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 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.

[0218] In some embodiments, each substituent described in the compounds herein is substituted with at least one substituent. More specifically, in some embodiments, each 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 described 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 substituent of limited size. In other embodiments, at least one or all of these groups are substituted with at least one low substituent.

[0219] In some embodiments, the compounds described herein may include multiple instances of substituents, for example, 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 8C In these embodiments, each substituent may optionally be different each time it occurs and be appropriately labeled to more clearly distinguish each group. For example, when each R 5A When different, they can be called, for example, R5A.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 Each of R can appear multiple times. 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 Each occurrence of is defined using 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 Definition of .

[0220] In one aspect, a compound of formula (II) is provided:

[0221]

[0222] or a pharmaceutically acceptable salt thereof. In formula (II), z1 is an integer from 0 to 4, z2 is an integer from 0 to 5, and R 5 is a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocycloalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group, R 6is 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 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.

[0223] In one embodiment, R 5 YesR 5A -substituted or unsubstituted cycloalkyl, R 5A -substituted or unsubstituted heterocycloalkyl, R 5A - substituted or unsubstituted aryl, or R 5A -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 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.

[0224] In addition, for this embodiment, R 6 YesR 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 -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 R 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 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.

[0225] In addition, for this embodiment, R 7 is hydrogen, or R 7A - substituted or unsubstituted alkyl. 7A 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.

[0226] In addition, for this embodiment, R8 R is independently halogen, -CN, -SH, -OH, -COOH, -NH2, -CONH2, nitro, -CF3, -CCl3, 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, R 8C - 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 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.

[0227] In another aspect, there is provided a compound of formula (II) as disclosed above, provided, however, that: (i) the compound of formula (II) is not

[0228]

[0229] Where R 5 is p-fluorophenyl or p-methylphenyl; (ii) the compound is not

[0230]

[0231] Where R 6 is unsubstituted aryl, unsubstituted cyclohexyl, unsubstituted thiazole or -CH2-furanyl; or (iii) R 7 Not hydrogen.

[0232] Further to any aspect disclosed above, in one embodiment, R 5 is not a substituted phenyl group. In one embodiment, R 5 Not p-fluorophenyl or p-methylphenyl.

[0233] In one embodiment, the compound does not have the structure of formula (IIa), wherein R 6 In one embodiment, the compound does not have the structure of formula (IIa), wherein R 6 It is p-fluorophenyl or p-methylphenyl.

[0234] Further to any aspect disclosed above, in one embodiment, R 6 is not a substituted or unsubstituted aryl, an unsubstituted cyclohexyl, an unsubstituted thiazole or a -CH2-furanyl. In one embodiment, the compound does not have a structure of formula (IIb), wherein R 6 is a substituted or unsubstituted aryl, a substituted or unsubstituted cyclohexyl, a substituted or unsubstituted thiazole, or a substituted or unsubstituted furanyl-substituted alkyl. 6 It is not unsubstituted aryl, unsubstituted cyclohexyl, unsubstituted thiazole or -CH2-furanyl.

[0235] Further to any aspect disclosed above, in one embodiment, R 5 is a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted aryl. 5 is an unsubstituted cycloalkyl group or an unsubstituted aryl group.

[0236] In one embodiment, R 5 is a substituted or unsubstituted C6-C8 cycloalkyl or a substituted or unsubstituted phenyl. 5 It is a substituted or unsubstituted C6 cycloalkyl group or a substituted or unsubstituted phenyl group.

[0237] In one embodiment, R 5 YesR 5A -substituted or unsubstituted C6 cycloalkyl or R 5A -substituted or unsubstituted phenyl, wherein R 5A is halogen. In one embodiment, R 5 YesR 5A-substituted or unsubstituted phenyl, wherein R 5A is halogen. In one embodiment, R 5 YesR 5A -substituted or unsubstituted phenyl, wherein R 5A In one embodiment, R 5 It is an unsubstituted phenyl group.

[0238] Further with respect to any aspect disclosed above, in one embodiment, the compound does not have a structure of Formula (Ib), wherein R 6 is a substituted or unsubstituted aryl group, a substituted or unsubstituted cyclohexyl group, a substituted or unsubstituted thiazole group, or an alkyl group substituted by a substituted or unsubstituted furyl group.

[0239] In one embodiment, R 6 is substituted or unsubstituted C4-C 12 Cycloalkyl, substituted or unsubstituted C3-C 12 alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In one embodiment, R 6 is substituted or unsubstituted C4-C 12 Cycloalkyl, substituted or unsubstituted C4-C 12 alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In one embodiment, R 6 is substituted or unsubstituted C4-C 12 Cycloalkyl, substituted or unsubstituted C4-C 12 branched alkyl, or substituted or unsubstituted phenyl. In one embodiment, R 6 YesR 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, wherein R 6A is halogen. In one embodiment, R 6 YesR 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, wherein R 6A In one embodiment, R 6 is an unsubstituted C4-C 12 Cycloalkyl, unsubstituted C4-C 12 Branched alkyl, or R 6A -substituted or unsubstituted phenyl, wherein R6A In one embodiment, R 6 It is an unsubstituted C6-C 12 Cycloalkyl, unsubstituted C4-C 12 In one embodiment, R 6 It is an unsubstituted C6-C 10 In one embodiment, R 6 is an unsubstituted C6-C8 cycloalkyl. 6 It is an unsubstituted cyclohexyl group.

[0240] In one embodiment, R 7 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. In one embodiment, R 7 is hydrogen, methyl or ethyl. In one embodiment, R 3 In one embodiment, R 7 In one embodiment, R 7 It's hydrogen.

[0241] In one embodiment, z1 is 0, 1, 2, 3, or 4. In one embodiment, z1 is 0 or 1. In one embodiment, z1 is 0. In one embodiment, z1 is 1. In one embodiment, z2 is 0, 1, 2, 3, 4, or 5. In one embodiment, z2 is 1.

[0242] In one embodiment, R 8 are independently substituted or unsubstituted alkyl. 8 is independently substituted alkyl. 8 is independently unsubstituted alkyl. 8 is independently substituted or unsubstituted heteroalkyl. 8 is independently substituted heteroalkyl. 8 is independently unsubstituted heteroalkyl. 8 are independently substituted or unsubstituted aryl. 8 and R is independently substituted or unsubstituted heteroaryl.

[0243]

[0244] For Formula (IIc) (above), R 6 is a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group; R7 is a substituted or unsubstituted alkyl group. 6 is an unsubstituted cycloalkyl group, for example, a cyclohexyl group, a cycloheptyl group or a cyclooctyl group. 6 is an unsubstituted alkyl group, for example, 3,3-dimethylbutyl. In one embodiment, R 7 is an unsubstituted alkyl group. In one embodiment, R 10 It is an alkyl ester.

[0245] In another aspect, a compound of formula (IId) is provided:

[0246]

[0247] For formula (IId), L2 is a linking group, and B1 is a purine base or an analog thereof.

[0248] In one embodiment, L2 is a substituted or unsubstituted alkylene, or a substituted or unsubstituted heteroalkylene. In one embodiment, L2 comprises a water-soluble polymer. "Water-soluble polymer" refers to a polymer that is sufficiently soluble in water for use in the methods described herein under physiological conditions such as temperature, ion concentration, etc. known in the art. An exemplary water-soluble polymer is polyethylene glycol.

[0249] In one embodiment, the water soluble polymer is -(OC2CH2) m -, wherein m is 1 to 100. In one embodiment, L 2 A "cleavage element" is a chemical functional group that can undergo cleavage (eg, hydrolysis) to release the compound, optionally including a linking group L. 2 The residue of B 1 , optionally including a linking group L 2 of the residue.

[0250] Table 1

[0251]

[0252] Table 2

[0253]

[0254]

[0255]

[0256] Table 3

[0257]

[0258] Route and preparation

[0259] The composition of one or more antigens and one or more adjuvants and optionally another active agent or the composition of one or more antigens and the composition of one or more adjuvants can be administered by any suitable route of administration, particularly parenteral, for example, intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular or subcutaneous. The administration can be a single large dose injection, multiple injections, or as a short-term or long-term infusion. Implantable devices (for example, implantable infusion pumps) can also be used for specific preparations to be delivered parenterally in equal doses or different doses over time. For this parenteral administration, the compound (conjugate or other active agent) can be formulated into a sterile solution in water or other suitable solvents or solvent mixtures. The solution may contain other substances, such as salts, sugars (particularly glucose or mannitol) that make the solution isotonic with blood, buffers such as acetic acid, citric acid and / or phosphoric acid and their sodium salts, and preservatives.

[0260] The compositions of the present invention, alone or in combination with other active agents, can be formulated into pharmaceutical compositions and administered to mammalian hosts, such as human patients, in a variety of forms suitable for the selected route of administration (e.g., oral or parenteral, by intravenous, intramuscular, topical or subcutaneous routes).

[0261] Thus, the compositions, alone or in combination with another active agent (e.g., an antigen), can be combined with a pharmaceutically acceptable carrier (e.g., an inert diluent or an assimilable edible carrier) for systemic administration, such as oral administration. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be mixed directly with food in the patient's diet. For oral therapeutic administration, the compositions, optionally in combination with the active compound, can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, flakes, and the like. The compositions and preparations should contain at least 0.1% of the active compound. Of course, the percentage content of the compositions and preparations can vary, generally from about 2 to about 60% by weight of a given dosage form. The content of the conjugate and optionally other active compounds in the useful composition is the content required to obtain an effective dosage level.

[0262] Tablets, lozenges, pills, capsules, etc. may also contain the following ingredients; binders such as tragacanth, gum arabic, corn starch or gelatin may be added; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid, etc.; lubricants such as magnesium stearate; sweeteners such as sucrose, fructose, lactose or saccharin, or flavoring agents such as mint, wintergreen oil or cherry flavor. When the dosage form is a capsule, in addition to the above materials, a liquid carrier such as vegetable oil or polyethylene glycol may be included. Various other materials may be present in the form of a coating, or the physical form of the solid unit dosage form may be changed. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar, etc. Syrups or elixirs may contain active compounds, sucrose or fructose as sweeteners, methylparaben and propylparaben as preservatives, dyes and flavoring agents such as cherry or sweet orange flavor, etc. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the phospholipid conjugates, optionally in combination with another active compound, can be incorporated into sustained-release preparations and devices.

[0263] The compositions optionally in combination with another active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of antigens and adjuvants optionally in combination with another active compound or a salt thereof may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, triacetin and mixtures thereof and in oil. Under normal conditions of storage and use, these preparations contain preservatives to prevent microbial growth.

[0264] The pharmaceutical dosage form suitable for injection or infusion may include a sterile aqueous solution or dispersion or sterile powder containing the active ingredient, which is suitable for instant preparation of sterile injection or infusion solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final dosage form should be sterile, mobile and stable under manufacturing and storage conditions. The liquid carrier or vehicle can be a solvent or liquid dispersion medium, which includes, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides and their suitable mixtures. For example, appropriate fluidity can be maintained by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by using surfactants. Various antibacterial and antifungal agents can be used during storage, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. to prevent microbial action. In many cases, it is useful to include isotonic agents such as sugars, buffers, and sodium chloride. The extended absorption of injectable compositions can be achieved by using delayed absorption agents in the composition, such as aluminum monostearate and gelatin.

[0265] The desired amount of the compound and the other various ingredients listed above (as needed) are added to a suitable solvent, followed by filtration sterilization to prepare a sterile injectable solution. When a sterile powder is used to prepare a sterile injectable solution, one preparation method includes vacuum drying and freeze drying techniques, which will obtain a powder of the active ingredient plus any other desired ingredients present in the previously sterile filtered solution.

[0266] For topical administration, the antigen and adjuvant, optionally in combination with another active compound, can be applied in pure form, for example, when they are liquids. However, it is usually desirable to apply them to the skin as a composition or formulation in combination with a skin-acceptable carrier (which can be solid or liquid).

[0267] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silicon dioxide, aluminum oxide, and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol mixtures, in which the compounds of the present invention can be dissolved or dispersed in effective amounts, optionally with the aid of nontoxic surfactants. Adjuvants such as fragrances and antimicrobial agents can be added to enhance the properties for a given use. The resulting liquid composition can be applied from an absorbent pad, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump or aerosol sprayer.

[0268] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with the liquid carrier to form spreadable pastes, gels, ointments, soaps and the like for application directly to the skin of the user.

[0269] In addition, in one embodiment, the present invention provides various dosage forms of antigens and adjuvants for inhalation delivery, optionally in combination with another active compound. For example, the formulation can be designed as an aerosol used in devices such as metered dose inhalers, dry powder inhalers and nebulizers.

[0270] Examples of useful skin compositions for delivering compounds to the skin are known in the art; see, e.g., Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157), and Wortzman (U.S. Pat. No. 4,820,508).

[0271] Useful doses can be determined by comparing their in vitro activity with in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art; see, for example, U.S. Pat. No. 4,938,949. The ability of an adjuvant to act as a TLR agonist can be determined using pharmacological models well known in the art, including Lee et al., Proc.Natl.Acad.Sci.USA , 100 :6646(2003) public proceedings.

[0272] Typically, the concentration of the phospholipids, optionally in combination with another active compound, in a liquid composition (e.g., a lotion) is about 0.1-25% by weight, e.g., about 0.5-10% by weight. The concentration in a semisolid or solid composition (e.g., a gel or powder) will be about 0.1-5% by weight, e.g., about 0.5-2.5% by weight.

[0273] The active ingredient may be administered so as to achieve a peak plasma concentration of the active compound of about 0.5 to about 75 μM, e.g., about 1 to 50 μM, e.g., about 2 to about 30 μM. This may be achieved, for example, by intravenous injection of a 0.05-5% solution of the active ingredient, optionally in saline, or orally as a bolus containing about 1-100 mg of the active ingredient. The desired blood concentration may be maintained by providing a continuous infusion of about 0.01-5.0 mg / kg / hr, or by intermittent infusions containing about 0.4-15 mg / kg of the active ingredient.

[0274] The amount of antigen and adjuvant required for treatment, optionally in combination with another active compound or its active salt or derivative, varies not only with the specific salt selected, but also with the route of administration, the nature of the disease to be treated, and the age and condition of the patient, and will ultimately be determined by the attending physician or clinician. However, in general, a suitable dosage range is from about 0.5 to about 100 mg / kg body weight, such as from about 10 to about 75 mg / kg body weight / day, such as 3 to about 50 mg / kg recipient body weight per day, such as 6 to 90 mg / kg / day, such as 15 to 60 mg / kg / day.

[0275] The antigen and adjuvant, optionally in combination with another active compound, may conveniently be administered in unit dosage form; for example, containing from 5 to 1000 mg, conveniently from 10 to 750 mg, most conveniently from 50 to 500 mg of active ingredient per unit dosage form.

[0276] The desired dose may conveniently be in the form of a single dose or divided doses administered at appropriate intervals, for example, in the form of 2, 3, 4 or more sub-doses per day. The sub-dose itself may be further divided into, for example, multiple dispersed loosely spaced administrations; for example, multiple inhalations from an insufflator or multiple drops instilled into the eye. The dose, and possibly the frequency of administration, will also vary according to the age, weight, condition and response of the individual patient. In general, the total daily dose range of the active agent for the diseases described herein may be from about 50 mg to about 5000 mg, taken in a single or divided dose. In one embodiment, the daily dose range should be from about 100 mg to about 4000 mg, for example, about 1000-3000 mg, taken in a single or divided dose, for example, 750 mg of the compound is taken orally every 6 hours. This can achieve a plasma concentration of about 500-750 uM, which can effectively kill cancer cells. When managing patients, treatment should be started at a lower dose based on the patient's overall response.

[0277] Specific antigens include amino acids, carbohydrates, peptides, proteins, nucleic acids, lipids, bodily substances or cells such as microorganisms.

[0278] Certain peptides have from 2 to about 20 amino acid residues.

[0279] Another specific peptide has 10 to about 20 amino acid residues.

[0280] Specific antigens include carbohydrates.

[0281] Specific antigens are microorganisms. Specific microorganisms are viruses, bacteria, or fungi.

[0282] Specific bacteria include Bacillus anthracis, Listeria monocytogenes, Francisella tularensis, Salmonella or Staphylococcus. Specific Salmonella is Salmonella typhimurium or Salmonella enteritidis. Specific Staphylococcus includes Staphylococcus aureus.

[0283] 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.

[0284] The invention includes compositions comprising TLR4 agonist and TLR7 agonist phospholipid conjugates, optionally in combination with other active agents, which may or may not be antigens, such as ribavirin, mizoribine, and mycophenolate mofetil.

[0285] Exemplary Embodiments

[0286] In one embodiment, a method for enhancing a mammalian immune response is provided. In one embodiment, the method comprises administering to a mammal in need thereof a composition comprising an effective dose of a TLR4 agonist and a TLR7 agonist. In one embodiment, the composition is a liposome composition. In one embodiment, the composition comprises liposomes containing a TLR4 agonist and a TLR7 agonist. In one embodiment, the composition comprises liposomes containing a TLR4 agonist and a TLR7 agonist. In one embodiment, a TLR4 agonist and a TLR7 agonist are administered simultaneously. In one embodiment, the TLR4 agonist has a structure of formula (II). In one embodiment, the TLR4 agonist comprises 1Z105, 2B182c, INI-2004 or CRX601. In one embodiment, the TRL4 agonist is not 1Z105. In one embodiment, the TLR7 agonist has a structure of formula (I). In one embodiment, the liposome comprises PC, DOPC or DSPC. In one embodiment, the liposome comprises cholesterol. In one embodiment, the method further comprises administering one or more immunogens. In one embodiment, the immunogen is a microbial immunogen, such as one or more microbial proteins, glycoproteins, carbohydrates and / or lipopolysaccharides. In one embodiment, the microorganism is a virus, such as influenza or chickenpox, or a bacterium. In one embodiment, the microorganism is a parasite or a fungus. In one embodiment, the liposome comprises one or more immunogens. In one embodiment, the composition comprises one or more immunogens. In one embodiment, the mammal is a human. In one embodiment, the mammal is a rodent, a horse, a cow, a goat, a dog, a cat, a pig or a sheep. In one embodiment, the content of the TLR7 agonist is about 0.01 to 100nmol, about 0.1 to 10nmol, or about 100nmol to about 1000nmol. In one embodiment, the content of the TLR4 agonist is about 2 to 20umol, about 20nmol to 2umol, or about 2umol to about 100umol. In one embodiment, the ratio of TLR7 and TLR4 agonists is about 1:10, 1:100, 1:200, 5:20, 5:100 or 5:200. In one embodiment, the composition is administered by injection. In one embodiment, the liposome comprises DOPC and cholesterol.

[0287] In one embodiment, the immunogen is a cell, protein or spore. In one embodiment, the immunogen is administered before or after the composition is administered. In one embodiment, administration is effective to prevent microbial infection. In one embodiment, the composition is administered intranasally. In one embodiment, the composition is administered intradermally.

[0288] In one embodiment, a pharmaceutical preparation comprising liposomes, a TLR4 agonist and a TLR7 agonist is provided. In one embodiment, the liposomes include 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'-racemic-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](16:0PEG-2000PE), 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-glycero-3-phosphocholine (18:1-12:0NBD PC), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazole-4-yl)amino]lauroyl}-sn-glycero-3-phosphocholine (16:0-12:0NBD 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 content of the TLR7 agonist is about 0.01 to 100nmol, about 0.1 to 10nmol or about 100nmol to about 1000nmol. In one embodiment, the content of the TLR4 agonist is about 2nmol to 20umol, about 20nmol to 2umol, or about 2umol to about 100umol. In one embodiment, the ratio of TLR7 to TLR4 agonists 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) comprises

[0289]

[0290] Where R 11 and R 12 are each independently hydrogen or acyl, R 13is a negative charge or hydrogen, and m is 1 to 8, wherein the wavy line indicates the bonding position, wherein in the case of 12 The absolute configuration at the carbon atom of is R, S or any mixture thereof. In one embodiment, m is 1. In one embodiment, R 11 and R 12 In one embodiment,

[0291] R 3 The phospholipid comprises two carboxylates, each carboxylate comprising 1, 2, 3 or 4 sites of unsaturation, epoxidation, hydroxylation or a combination thereof. In one embodiment, R 3 The phospholipids of the invention comprise two carboxylates, which are similar or different. In one embodiment, each carboxylate of the phospholipids is a C17 carboxylate with an unsaturated site at C8-C9. In one embodiment, each carboxylate of the phospholipids is a C18 carboxylate with an unsaturated site at C9-C10. In one embodiment, X 2 can be a bond or a chain having from 1 to about 10 atoms in the chain, wherein the atoms of the chain are selected from carbon, nitrogen, sulfur and oxygen, wherein any carbon atom may be substituted by oxygen, wherein any sulfur atom may be substituted by one or two oxygen atoms. In one embodiment, X 2 is C(O),

[0292]

[0293]

[0294] In one embodiment, R 3 In one embodiment, R 3 is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, X 2 is C(O). In one embodiment, X 1 In one embodiment, X 1 is sulfur, or -NR c -, where R c It is hydrogen, C 1-6 Alkyl or substituted C 1-6 Alkyl, wherein the alkyl substituent is hydroxyl, C 3-6 Cycloalkyl, C 1-6 In one embodiment, X 1 is -NH-. In one embodiment, R 1 and R c Together they form a heterocyclic or substituted heterocyclic ring. 1 and R cTogether they form a substituted or unsubstituted morpholine, piperidine, pyrrolidine or piperazine ring. In one embodiment, R 1 is a C1-C10 alkyl group substituted with a C1-6 alkoxy group. 1 It is hydrogen, C 1-4 Alkyl or substituted C 1-4 In one embodiment, R 1 is hydrogen, methyl, ethyl, propyl, butyl, hydroxyl C 1-4 Alkylene or C 1-4 Alkoxy C 1-4 In one embodiment, R 1 is hydrogen, methyl, ethyl, methoxyethyl or ethoxyethyl. In one embodiment, R 2 Is halogen or C 1-4 Alkyl, or R 2 In one embodiment, R 2 is chlorine, bromine, methyl or ethyl, or R 2 In one embodiment, X 1 It's O, R 1 It is C 1-4 Alkoxy-ethyl, n is 0, X 2 is a carbonyl group, and R 3 is 1,2-dioleoylphosphatidylethanolamine (DOPE). In one embodiment, the compound of formula (I) is:

[0295]

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

[0297]

[0298] In one embodiment, in formula (II), z2 is 1, 2 or 3. In one embodiment, in formula (II), z1 is 1 or 2. In one embodiment, in formula (II), z1 is 0. In one embodiment, in formula (II), R 5 is a substituted or unsubstituted aryl or heteroaryl, for example, an unsubstituted C5 or C6 aryl. In one embodiment, in formula (II), R 6 is a substituted or unsubstituted cycloalkyl or heterocycloalkyl, such as a C5, C6 or C7 cycloalkyl. 7 is a substituted or unsubstituted alkyl group, such as a C1 to C5 alkyl group. In one embodiment, in formula (II), R 8 is substituted or unsubstituted aryl or heteroaryl, for example, C5, C6 or C7 heteroaryl, for example, furyl, pyrrolyl or imidazolyl.

[0299] The present invention will be further illustrated by the following non-limiting examples.

[0300] Example 1

[0301] Efficacy of liposomally formulated 2B182c (TLR4 agonist) and 1V270 (TLR7 agonist) adjuvants

[0302] Liposome preparations of separate 2B182c (200nmol / needle) and 1V270 (1nmol / needle) or liposome preparations of 200nmol2B182c and 1nmol 1V270 combination (Inimmune Corp, Missoula, Montana). The auxiliary efficacy of liposome-formulated adjuvants was compared with DMSO preparations (10% DMSO). The preparation adjuvants were tested using the same protocol. In brief, female BALB / c mice were immunized with inactivated influenza virus using liposome-formulated 2B182c (200nmol / needle) and / or 1V270 (1nmol / needle) at day 0 and day 21, and serum anti-HA and anti-NA antibodies (IgM, IgG1 and IgG2a) were assessed by ELISA. Inguinal lymph nodes were harvested, and the number of B cells was analyzed by FACS to investigate whether the agonist prepared affects the number of germinal center B cells and plasmablasts (antigen secreting cells).

[0303] TLR4 is located on the cell surface and in the endosome lumen. Signaling through endosomal receptors inhibits NF-κB activation by LPS. Endosomal TLR4 activation triggers TRIF pathway activation, leading to type 1 IFN release through IRF3 activation. Therefore, liposomal formulations may attenuate the adjuvant activity of 2B182c. When mice were immunized with 2B182c alone or with 2B182c+1V270, liposomal 2B182c induced significantly higher anti-HA IgG2a compared with DMSO-formulated adjuvant, while liposomal 2B182c reduced HA- and NA-specific IgG1( Fig.19 A). Liposome formulation did not affect the IgG2a levels of 2B182c and 2B182c+1V270 combination adjuvants ( Fig.19 A). The liposome formulation reduced IgG1 levels because the liposome-formulated 2B182c and 2B182 / 1V270 combination adjuvants induced a Th1-biased immune response ( Fig.19 B) These data are consistent with reports that intracellular delivery of TLR4 ligands induces potent Th1 immune responses in a type 1 IFN-dependent manner.

[0304] After exposure to antigen, activated naive and memory B cells expand and mature in germinal centers (GCs). High antigen-specific antibody titers required to maintain long-term vaccine efficacy are associated with GC formation. Activated B cells further form antigen-specific antibody-secreting cells (ASCs; plasmablasts and plasma cells), memory B cells, and other subsets. Plasmablasts are induced after seasonal influenza virus vaccination and peak at day 7 after vaccination. The frequency of plasmablasts in peripheral blood after vaccination with inactivated virus correlates with the magnitude of protective hemagglutinin inhibition titers in humans. Therefore, GC B cells and plasmablasts in draining lymph nodes were measured. The number of germinal center B cells and plasmablasts was increased by a combination of liposomes 2B182c and 1V270 ( Fig. 20 ).

[0305] In conclusion, liposomally formulated TLR4 and TLR7 ligand adjuvants induced a Th1-biased immune response and increased the number of B cells and plasmablasts in the GC center. To evaluate the quality of the B cell response induced by the combined adjuvants in the liposomal formulation, we are currently performing BCR and TCR repertoire analysis on lymph node cells. In addition, functional evaluation of vaccine adjuvants was assessed by live virus (homologous and heterologous challenge).

[0306] Example 2

[0307] Combinations of synthetic small molecule TLR4 and TLR7 agonists are effective adjuvants to recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity and protecting against influenza virus in homologous, heterologous, and heterosubtypic mouse challenge models. However, the TLR4 agonist used in these studies was 1Z105, a first-generation lead synthetic TLR4 agonist in the pyrimidoindole class that was derived from optimization of hits identified from high-throughput screening campaigns to discover adjuvants that act as innate immune receptor agonists. 1Z105 was found to have good immunological activity in murine cells but no significant activity in human cells. In recent studies, a second-generation series of compounds containing C8-aryl substituents were more potent than 1Z105 in murine cells but also very 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 preliminary formulation data ( Fig.34 and 36 ). The pyrimidoindole 2B182C was evaluated in combination with 1V270 for comparison. The MPLA analog (MPLA-1) is a potent TLR4 agonist that exhibits good protection against homologous and heterologous influenza challenges in vivo.

[0308] The TLR7 agonist 1V270 is a phospholipid conjugate of a known TLR7 agonist. The major advantages provided by the phospholipid portion of the agonist conjugate relative to the corresponding unconjugated agonist include greater potency and the absence of local or systemic toxicity typically observed as cytokine syndrome. These favorable properties of efficacy and safety support the selection of 1V270 as the primary TLR7 agonist for the combined adjuvant studies described in this technical solution.

[0309] As previously described, a combination adjuvant comprising the TLR4 agonist 1Z105 and the TLR7 agonist 1V270 induces a broad range of protective responses in influenza virus vaccines. 2B182C from SAR studies showed higher agonist efficacy than 1Z105 in THP-1 cells and in vitro human and mouse primary cells. The adjuvant efficacy of 2B182C was investigated in a vaccination model using inactivated influenza virus [A / California / 04 / 09 (Cal / 09)] and compared with 1Z105. These studies were performed using simple DMSO-water formulations of TLR agonists.

[0310] Combination adjuvants of TLR4 and TLR7 agonists induce rapid and broad protection against influenza virus infection Sexual immune response

[0311] To evaluate the profile of TLR4 / TLR7 agonist combination adjuvants in inducing protective immune responses against influenza virus infection, mice were immunized with a low dose (0.2 μg / injection) of recombinant hemagglutinin (rHA) to examine humoral responses and protection against lethal virus challenge ( Fig.33 A-33D). Mice immunized with rHA and combined adjuvants showed minimal weight loss and higher survival rate ( Fig.33 B-33C). Both the combined adjuvant and the TLR7 agonist 1V270 alone induced a Th1-biased immune response.

[0312] To further investigate whether the TLR4 / TLR7 combination adjuvant provides cross-protection against heterotypic influenza virus challenge, we immunized mice with 2009-2010 Fluzone containing B / Brisbane / 60 / 2008 (Victoria lineage) and challenged them with 25 mLD50 of the heterologous mouse-adapted virus B / Florida / 04 / 2006 (Yamagata lineage). After vaccination with Fluzone adjuvanted with 1V270 alone or in combination with 1Z105, more than 90% of the mice survived ( Fig.33 These data suggest that 1V270 alone or in combination with 1Z105 can induce rapid cross-protective immunity against heterologous influenza viruses.

[0313] Determining the dose of TLR4 and TLR7 agonists

[0314] As previously described, 2B182C was derived from SAR studies and showed higher potency in vitro compared with 1Z105 in human and mouse immune cells. To investigate whether the higher potency observed in vitro could also be reproduced in vivo, female Balb / c mice were challenged with activated influenza virus (A / California / 04 / 2009(H1N1)pdm09, Cat # Intramuscular (IM) immunization was performed with TLR4 agonists (1Z105 or 2B182C, 40 or 200 nmol / injection) and 1V270 (phospholipid TLR7 agonist conjugate, 0.2 or 1 nmol / injection) in 1:1% NR-49450, BEI Resources Fig.34 A). Serum was collected on day 28 and antibodies (IgM, IgG1 and IgG2a) against hemagglutinin (HA) and neuraminidase (NA) were determined by ELISA. 1V270, 1Z105 and 2B182C were dissolved in DMSO and diluted to a final concentration of 10% in DMSO and used as vehicle control. Data are pooled from four independent experiments showing similar results.

[0315] Effects of single adjuvant of TLR agonist on antibody secretion

[0316] The results were compared when 0.2 nmol and 1 nmol 1V270, and 40 nmol and 200 nmol 2B182C or 1Z105 were used as single adjuvants ( Fig.34 B). Both TLR4 agonists 1Z105 and 2B182C induced significantly higher levels of IgG1 against HA and NA. Regarding IgG2a induction, both 0.2 and 1 nmol / injection 1V270 significantly increased anti-HA (p<0.05), while only 200 nmol / injection 2B182C, but not 1Z105, enhanced anti-NA Abs (p<0.01) ( Fig.34 B). 2B182C and 1Z105 induced similar levels of HA-specific IgG2a. There were no differences in IgM responses elicited by any of the adjuvant treatments. These data support reports that TLR4 agonists increase IgG1 production, TLR7 agonists are effective on IgG2a secretion, and 2B182C exhibits similar or modestly higher potency than 1Z105 in vivo.

[0317] Effect of 2B182C and 1V270 combination treatment on antibody secretion

[0318] Next, the efficacy of the combination adjuvants was evaluated using a DMSO-water formulation. Both combination adjuvants, 1V270 combined with 1Z105 and 2B182C, improved the induction of anti-HA and anti-NA IgG1. Compared with 1Z105, 2B182C significantly increased IgG1 at both 40 and 200 nmol (p<0.05, Fig.35 A and 35B). In IgG2a induction, 2B182C increased the levels of anti-HA- and anti-NA-Abs; however, 1Z105 failed to increase their levels in most cases ( Fig.35 C and 35D). The adjuvant had little effect on IgM release ( Fig.35 E and 35F).

[0319] To compare the antibody titers of all tested combinations, the mean IgG1 and IgG2a titers were plotted on Fig.36 In A, 200 nmol 2B182C + 0.2 or 1 nmol 1V270 showed the highest induction for IgG1 and IgG2a ( Fig.36 A). In addition, to assess the Th1 / Th2 immune balance, the IgG2a:IgG1 ( Fig.36 B). 1 nmol 1V270 significantly altered the Th2-biased immune response by 1Z105 or 2B182C, indicating that 1V270 shifted the immune response to Th1 bias ( Fig.36 B). In summary, these results indicate that the combination of 200 nmol / injection 2B182C + 1 nmol / injection 1V270 induced the highest amounts of IgG1 and IgG2a, and that a Th1-biased immune response is desirable for heterologous protection in influenza virus infection. Therefore, we selected this combination for further preclinical formulation.

[0320] Preliminary data on TLR4 agonists

[0321] MPLA-2 (a sulfate analog of MPLA), combinations, and all major TLR agonists in nanoparticle formulations were evaluated in vivo. A potent TLR4 agonist was discovered during an NIAID adjuvant discovery and development contract that demonstrated additive, if not synergistic, enhancement of influenza-associated cytokine production in vitro (in hPBMCs), enhanced IgG2A antibodies and HI titers in mice and pigs with 1V270. A major weakness of MPLA-1 as an adjuvant is its easy hydrolysis in aqueous media and lack of chemical stability. In preliminary murine studies of nonspecific resistance, MPLA-2 protected mice better from lethal influenza challenge than an equivalent dose of MPLA-1, and therefore, MPLA-2 represents the next generation of TLR4 agonists.

[0322] In support of the goals outlined above, the experiments detailed below were performed.

[0323] Research Area 1: Formulation and Analytical Assay Development for Major TLR Agonist Combinations

[0324] Development of TLR4 / TLR7 combination agents

[0325] Task 1A: Development of colloidally stable nanoparticle formulations of lead compounds alone and in combination

[0326] Microparticle delivery systems act as adjuvants by mimicking the size and shape of viral and bacterial pathogens, which our immune system has evolved to recognize and combat via pattern recognition receptors (PRRs). Research over the past 30 years has developed a number of nano- and microparticle-based systems that are biodegradable and suitable for vaccine antigen delivery. Their utility as vaccine delivery systems has been demonstrated in the literature, including liposomes, virosomes, immunostimulatory complexes, emulsions, virus-like particles (VLPs), solid lipid nanoparticles (SLNs), and polylactic-co-glycolic acid (PLGA) polymers, with examples of each type entering human clinical trials. The primary adjuvant mechanism of microparticle delivery vehicles is thought to be enhanced uptake of particles incorporated or bound to antigen by APCs. It is now established that the addition of PAMPs to antigens can cause activation of innate immune cells by ligating TLRs and other PRRs, promoting strong innate and adaptive immune responses. Many PAMPs (bacterial lipoproteins, glycolipids, DNA, and viral RNA, among others) have been identified and isolated from viral and bacterial pathogens. Many of these agonists are powerful adjuvants but produce 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 properties, many of which have been added to microparticle delivery systems to enhance their pathogen mimicry via PRR attachment. Microparticle delivery systems can also be used to improve the in vivo biodistribution kinetics of adjuvants and reduce adjuvant side effects without sacrificing adjuvant immunogenicity.

[0327] In a mouse model of influenza, the effective use of bilayered PEGylated liposomes containing the TLR4 agonist MPLA-1 as a sublingual vaccine has been demonstrated. This formulation reduced the pyrogenicity of MPLA-1 by 200-fold without any loss of adjuvant potency in vivo. This is similar to the reduction in pyrogenicity observed for LPS when incorporated into liposomes compared to aqueous dispersions. It is expected that the pyrogenicity of TLR4 agonists will be similarly reduced.

[0328] A number of different lipids and compositions were evaluated for nanoparticle / microparticle formation, API incorporation, API stability, and colloidal stability. A range of commercially available cationic (DDA, DOTAP, DC-cholesterol), anionic (DPPG, PS, POPG), and neutral lipids (PC, DOPC, DSPC) were tested with TLR4 and TLR7 agonists. Other formulations may use PLGA, polycaprolactone, poly(propargyl methacrylate), or PLMA. Because particle size and charge have been shown to significantly affect nanoparticle uptake and processing by DC, the impact of these variables on delivery vehicle design to enhance the above quality properties was explored. Small quantities of liposomal formulations can be prepared using a thin-film method suitable for sterile serum bottles to further reduce scale and waste.

[0329] In short, this will be done by:

[0330] 1. Add the API to the lipids and dissolve them in chloroform (if desired, a fluorescent label such as NBD, BODIPY, FITC, etc. can also be added at this step).

[0331] 2. Rotate and evaporate at set speed and vacuum to a dry film.

[0332] 3. Rehydrate with aqueous buffer (0.1 M phosphate, TRIS or HEPES).

[0333] 4. Particle size reduction by bath sonication above the lipid transition temperature (Tm) and in-process monitoring of particle size, polydispersity and surface charge (ζ-potential) by dynamic light scattering (DLS).

[0334] 5. 3 to 10 mL scale batches of the lead formulation were prepared using a Lipex extruder, which improved particle size uniformity (polydispersity index, PDI) compared to the sonication method.

[0335] Task 1B: Stability studies to evaluate the colloidal and physical stability of the formulation

[0336] The development of successful commercial products requires formulation stability as it affects the storage, transportation and shelf life of the product, which directly impacts product cost. These formulations are proven to be suitable as potential products, as well as stable, especially when selecting lead candidates for further testing.

[0337] The lead formulation is 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 adequate stability for potential product development (preferably at least 12 months under storage conditions).

[0338] Accurate and quantitative incorporation of adjuvants into nanoparticle delivery systems is critical for correct dosing, vaccine efficacy, and safety. SEC-HPLC and RP-HPLC methods have been developed for quantification of TLR4 and TLR7 / 8 agonists incorporated into nanoparticles (including liposomes). RP-HPLC can effectively analyze the total agonist content present in nanoparticles when the sample is dissolved in a water-miscible organic solvent (methanol, tetrahydrofuran, etc.) with sufficiently low background UV absorption. Dissolution with organic solvents will destroy the nanoparticles and release any incorporated or surface-bound agonists, which are accurately quantified by RP-HPLC against a 5-point standard curve.

[0339] For quantification of liposome-incorporated (bilayer or aqueous core) agonists, a method capable of analyzing both intact liposomes and the extraliposome aqueous phase is required. A SEC-HPLC method capable of quantifying “free” TLR agonists by UV detection at 296, 225, and 310 nm (for 2B182C, MPLA-2, and 1V270, respectively) was employed. TSK gelSWxl series columns provide excellent size-based separations for nanoparticle formulations in the 30–200 nm range. The mobile phase used was the same buffer used for rehydration of the liposomes to maintain a constant osmotic potential between the extraliposome fluid and the aqueous phase in the liposome core. This method can be used as a complement to in vitro potency assays that only test aqueous, unincorporated TLR4 agonists.

[0340] Initial studies were conducted using these analytical methods to evaluate liposomal formulations of 2B182C and 1V270, each prepared individually and in combination (co-encapsulation). The workflow was as follows:

[0341] 1) 2 mL scale lead adjuvant formulation screening (pharmaceutically acceptable co-solvents, excipients, liposomes) with a target concentration of 1 nmol 1V270 and 200 nmol 2B182C contained in 50 uL for intramuscular injection (IM).

[0342] 2) Perform basic analytical method development and analysis on the lead product to ensure that the product meets the quality standards.

[0343] 3) Evaluate the stability of the preferred formulation by real-time and accelerated methods and add appropriate excipients as stabilizers if necessary.

[0344] 4) Conduct rFC testing to ensure that the finished dosage form is free of endotoxin contamination.

[0345] For compounds 2B182C and 1V270, all liposome formulations were prepared at 2 mL scale.

[0346] In brief, liposomes were prepared using the following procedure and the following compositions were evaluated: 2B182, in combination with and without 1V270 (with DOPC / with and without cholesterol, 2:1, respectively). The DOPC concentration tested was kept constant at 40 mg / mL, which resulted in a cholesterol concentration of 10 mg / mL. Liposomes were produced according to the lipid film rehydration method using 9:1 chloroform:methanol as solvent. The rehydration buffer initially used was 50 mM NaBP, 100 mM NaCl, pH=6.1. The agonist concentration tested was the target concentration. Ultrasonication at higher temperatures reduced the liposome particle size. A summary of the analytical results is shown in Table 4.

[0347]

[0348]

[0349] Table 5. Analysis of preliminary liposomal formulations of 2B182C and 1V270 demonstrated comprehensive analytical characterization of the lead formulations.

[0350] Other TLR agonist ratios, other lipid components, and different amounts of cholesterol were evaluated for nanoparticle formation. At least 10 different formulations were prepared and screened for suitability during Task 2A and compared to the results we obtained using the simple DMSO–water formulation described above.

[0351] 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 production data. Based on preliminary immunology and stability data, DOPC / cholesterol liposome formulations seem very promising. One of the expected challenges of TLR4 and TLR7 agonists is that the two agonists are co-incorporated into the same nanoparticle in a controlled and consistent manner. Once co-encapsulated, the ratio of the agonists to each other is fixed, so any dosage adjustment at this time will change the two agonists at the same time.

[0352] Analytical methods: All analytical methods described in Task 1C have been used for our lipidated TLR-7 / 8 agonists and TLR4 agonists, and we hope to further improve their specificity, linearity, and range through further optimization. The RP-HPLC methods used to quantify adjuvants in TLR4 and TLR7 agonist formulations will be optimized for peak shape, LOD, and LOQ. These same methods will be gradient- and column-optimized to achieve baseline separation and optimal LOD / LOQ for any degradants detected in stability studies, thereby enabling accurate monitoring of product stability. Accurate quantification of the percent nanoparticle incorporation of each agonist in the TLR4 and TLR7 agonist combination may be challenging because SEC-HPLC separates based only on hydrodynamic volume. Liposomes and unincorporated agonist may have similar particle sizes, which will limit the practicality of SEC-HPLC for incorporation determinations. This will be discussed in the alternative approach below.

[0353] Alternative method : If the development of co-encapsulated TLR4 and TLR7 agonists proves too difficult due to inconsistent agonist levels in the nanoparticles, our immunological data suggest that adjuvant synergy may still be achieved by simply mixing a TLR4 agonist in liposomes with a TLR7 agonist in liposomes. This approach has the potential to produce a simpler, more robust product that can be more easily characterized analytically by reducing the potential for interference between agonist signals.

[0354] Another option is to explore other co-encapsulation formulations, such as nanoemulsions, in which the agonist is 100% incorporated by default because the aqueous and oil phases mix to form nanodroplets. Emulsions also have the advantage of forming a depot at the site of administration, which can further enhance the immune response. As discussed in Research Area 2, co-encapsulated TLR4 and TLR7 agonists are compared to mixed TLR4 and TLR7 agonists in vitro and in vivo to weigh the pros and cons of these approaches. Another approach to determine the agonist incorporated into the nanoparticles using SEC-HPLC is to pellet the nanoparticles using high-speed density gradient centrifugation and analyze the unincorporated agonist in the supernatant using an established RP-HPLC method.

[0355] Formulations with acceptable improved properties within the target ratio range were subjected to in vivo studies, including immunization and viral challenge studies.

[0356] Research Area 2: Identifying lead adjuvant formulations that protect against lethal influenza virus challenge

[0357] Immunological biomarkers

[0358] The successful development of safe and effective vaccines requires the definition of reliable biomarkers. For vaccine development programs, it is crucial to select candidate vaccines that effectively prevent infection and do not have any safety issues. In vaccine clinical trials, it is necessary to identify biomarkers that predict antigen-specific adaptive immune responses with minimal reactogenicity. In this project, the identification of biomarkers was carried out in two steps: 1) identification of innate immune biomarkers induced by formulated lead adjuvants with and without antigens, and 2) biomarkers associated with adaptive immune responses. Therefore, in vitro and in vivo studies were performed to identify biomarker candidates that correlated with the biological activity of TLR4 and TLR7 / 8 ligands and also correlated with reactogenicity.

[0359] Task 2A: Study of immunological activity and reactogenicity of combination preparations based on in vivo antibody production

[0360] The hallmark of preventing infectious diseases through vaccination is the induction of effective antibody production. The combination of TLR4 and TLR7 agonists resulted in a significant increase in antigen-specific antibody titers. A Th1 bias in the immune response was observed. The effectiveness of the formulated adjuvants and their combination was compared with a simple DMSO-water formulation.

[0361] Task 2A.1: Mouse Immunization Study of Lead Combination Formulation

[0362] The lead adjuvant formulation was evaluated in an immunization study of separate and different ratios of TLR agonist combinations using a similar approach previously completed for the DMSO-water formulation. Levels of IgM, total IgG, and IgG1 and IgG2a specific for HA and NA were evaluated. One or more ratios of TLR agonist combinations that provided the maximum titer of antigen-specific antibodies were identified. This formulation will be further used in challenge studies in Research Area 3.

[0363] Task 2A.2: Reactogenicity and toxicity evaluation of the lead combination formulation in mice

[0364] Because infectious disease vaccines are intended to provide protection to healthy individuals, vaccine safety must be the highest priority among development goals. Therefore, appropriate experiments are performed to evaluate the toxicity and reactogenicity of candidate agents. In these experiments, in general, overt toxicity is closely evaluated as an initial toxicity assessment. Any signs of distress in mice (i.e., unkempt hair, reduced mobility, conjunctiva , abnormal behavior, responsiveness, etc.). In addition to gross observations, toxicity assays included complete blood counts, serum chemistry assessments (AST, ALT, ALP, amylase, blood urea nitrogen, creatinine, total protein, glucose, potassium, calcium, sodium, total bilirubin), and necropsy evaluations (spleen, liver, and kidney sections stained with hematoxylin and eosin). In addition, the injection site was evaluated for visible signs of inflammation and any other abnormal findings. Tissues from the injection site were also evaluated histologically as part of the necropsy evaluation. The results of the study are summarized in Table 6 below.

[0365] Task 2B: Identification of immune markers that predict protective adaptive immune responses

[0366] As mentioned above, identification of biomarkers that predict antigen-specific adaptive immune responses with minimal reactogenicity could aid clinical trial design and methodology.

[0367] Task 2B.1: Characterization of innate immune responses (cytokines, chemokines)

[0368] Recruitment of immune cells to the local site of vaccine administration by chemokines is critical for recruiting antigen presenting cells (APCs) and influencing the induction of subsequent adaptive immune responses. However, the injection site, muscle tissue, contains relatively few 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 is able to express sufficient chemokines to recruit inflammatory cells. Following TLR stimulation, it is difficult to distinguish inflammatory responses from adjuvant effects because the recruitment of APCs is often accompanied by inflammatory cells. These complex immune activation cascades cannot be studied in vitro assays alone. Therefore, the marker panel was selected from the above in vitro experiments with samples obtained from in vivo studies in mice.

[0369] The lead adjuvant formulation was injected intramuscularly (IM) into mice, and sera were collected on days 1, 3, and 7 post-injection for systemic cytokine / chemokine levels. As described above in Task 2A.2 for local muscle tissue, cytokine / chemokine and co-stimulatory molecule gene expression will be investigated by qPCR or NanoString analysis. Selected samples were examined histologically by hematoxylin-eosin staining and immunohistochemical staining to assess immune cell infiltration. Splenocytes or PBMCs were used to assess the expression of co-stimulatory molecules by flow cytometry. Draining lymph nodes were collected at the indicated time points and pooled into each experimental group and analyzed for immune cell numbers and expression of chemokine receptors and co-stimulatory molecules. A summary of the study design is shown in Table 6. Note that "Group 5: Combination Adjuvant and Antigen Group" can include combinations of TLR4 and TLR7 agonists in different ratios as needed to provide the desired cytokine / chemokine induction characteristics. Innate immune features that show TLR4 and TLR7 ligand bioactivity and also correlate with reactogenicity were selected.

[0370] Table 6

[0371]

[0372] Task 2B.2: Characterization of the adaptive immune response.

[0373] Experiments to assess adaptive immune responses were performed in conjunction with Task 2A.1 above. Candidate biomarkers were identified that met the following criteria: 1) detectable in peripheral blood, 2) driven by the mechanism of action of each TLR ligand and associated with its biological effect, 3) predictive of long-term antigen-specific antibody induction and broad protection, and 4) predictive of reactogenicity.

[0374] Results and alternative approaches

[0375] Combinations of TLR agonists in one or more ratios provide the greatest titer of antigen-specific antibodies. In addition, the use of a combination of two classes of TLR ligands results in a shift in the adaptive immune response toward a Th1-biased response compared to the use of a TLR4 agonist alone. As a result, the Th1 / Th2 response ratio may be increased. This Th1 bias may be beneficial for expanding the response to include heterologous virus protection. As for toxicity, systemic and oral administration of imidazoquinoline TLR7 / 8 ligands has shown severe side effects, including flu-like symptoms, nausea and lymphopenia, as well as high levels of serum TNFα and IL-1β. This is also the case for the oxoadenine class, of which 1V270 is a member. However, vaccination using the usual local injection route of administration, i.e., intramuscular injection, can avoid most of these undesirable side effects. In addition, TLR7 / 8 ligands are prepared by conjugation to lipid moieties and customized formulations, successfully reducing systemic cytokine release while maintaining adjuvant activity. Therefore, due to low systemic exposure to inflammatory cytokines, there may be little or no reactogenicity associated with the lead formulation combination.

[0376] Research Area 3: Selection of Agents and Immunization - Mouse Virus Challenge Studies (Immunization)

[0377] Based on the results of formulation studies including stability (Task 1B), immunological activity (Task 2A.1) and reactogenicity characteristics (Task 2A.2), the lead formulation combination and the backup combination are selected for preclinical immunization / viral challenge studies in mice. The viral antigens used for the study can be selected from recombinant vaccine antigens or inactivated whole viruses that have been used in licensed commercial vaccines, such as A / Victoria / 3 / 75(H3N2), A / Michigan / 45 / 2015(H1N1)pdm09-likevirus and A / Hong Kong / 4801 / 2014(H3N2)-like viruses.

[0378] Task 3A: Selection of lead combination formulations

[0379] Lead selection criteria were based on: 1) stability of the formulated combination, 2) ratio of TLR agonists that provided the desired antigen-specific antibody levels, and 3) low local and systemic reactogenicity profiles. Specific studies relevant to these criteria are summarized in Table 7.

[0380] After the lead formulation combination and alternate lead combinations are selected, the selections will be evaluated in a mouse immunization / viral challenge model (Task 3B).

[0381]

[0382]

[0383] Table 7. Summary of assay results

[0384] Task 3B: Immunization / Viral Challenge Studies Using Lead Agents

[0385] Task 3B.1: Determine the Minimum Protective Dose for Virus Challenge Studies

[0386] Since inactivated influenza viruses contain innate immune receptor ligands (PAMPS), it is expected that some low level of protection may occur after immunization of mice with only sufficient antigen. Therefore, a study was conducted to determine the lowest protective dose (if any) of inactivated virus. The lowest protective dose of antigen is the dose that only provides partial protection (less than 30% survival) when subsequently challenged with a matched active virus strain. This strategy allows a range of activity to be observed using the selected lead formulation adjuvant combination. In addition, it is also possible to confirm the amount of challenge virus that causes total mortality of non-immunized mice, usually a dose of about 5LD50.

[0387] Task 3B.2: Homologous virus protection studies

[0388] Following antigen dose-range finding studies, mouse models were used to evaluate the immunogenicity of lead adjuvant combinations as well as homologous influenza vaccine antigens. The main determinants of success were: 1) persistent influenza-specific IgG2a and IgG1 in serum, 2) protection against lethal influenza virus challenge, 3) low reactogenicity, and 4) induction of polyfunctional CD4+ and CD8+ T cells assessed by intracellular IFNγ / TNFα staining. Secondary endpoints included weight gain / loss and scoring of disease severity by monitoring observable clinical symptoms (ruffled fur, arched back, and difficulty breathing) after vaccination or influenza virus challenge.

[0389] General in vivo methods

[0390] Immunological evaluation: Mice (male and female) were vaccinated intramuscularly (adjuvant + influenza antigen, e.g., A / Victoria / 3 / 75(H3N2)) 1 to 2 times with an interval of 21 days between the first and second vaccinations. Fig.12). Cell-mediated immunity (CMI) was assessed in a subset of 4 mice per group by measuring Th1 / Th2 cytokine induction in splenocyte cultures (determined by ELISA) and polyfunctional CD4+ and CD8+ T cell responses (determined by FACS, 10-color intracellular cytokine staining). In addition, tetramer staining and cell surface phenotyping were performed to determine the frequency of influenza-specific memory CD4+ and CD8+ T cells. Influenza-specific humoral responses were measured in serum (IgG1 and IgG2a), and HI titers were used to measure functional antibody titers. Vaccinated mice and control mice were challenged with 5LD50 of A / HK / 68(H3N2) and survival, weight gain / loss, and disease severity scores were assessed over 21 days. Reactogenicity in these mouse studies was measured by weight loss and symptom scores and injection site infiltration assessments. Differences in p values ​​<0.05 were considered significant. All data were analyzed by analysis of variance (ANOVA) and Tukey ANOVA to demonstrate robust statistical significance.

[0391] Task 3B.3: Heterologous Virus Protection Studies

[0392] Following homologous protection studies, lead adjuvant combinations were evaluated in a heterologous or heterosubtypic protection mouse model using the same study design. Mice were immunized as described above (Task 3B.2), but were challenged with an influenza virus strain of a different HA / NA type (e.g., A / Puerto Rico / 8 / 1934 (H1N1)). The protection observed in this challenge model suggests that the antigen-specific response was expanded to include antigens common to both strains, such as the stem region of the HA protein. To confirm this expansion, studies of B cell receptor (BCR) and T cell receptor (TCR) sequences were performed.

[0393] Results and alternative approaches

[0394] As mentioned above, there is an increasing number of reports in the literature listing various combinations of TLR agonists that can synergistically increase the strength of vaccine-mediated immunity and alter the type of downstream adaptive immune response generated, thereby improving the efficacy of these vaccines. This article describes adjuvant combinations for protection against influenza virus challenge.

[0395] Example 3

[0396] Strategies to enhance broadly neutralizing stalk antibodies using influenza hemagglutinin (HA) as a vaccine antigen include: 1) focusing on headless HA, removing the entire head domain to make the stalk domain more “available” and thereby inducing antibody responses against the stalk domain, or 2) using chimeric HA composed of stalk domains from a combination of H1, H3, or B influenza viruses.

[0397] It is well known that immunization with one antigen blocks a strong immune response to a second, similar antigen ("traces of the original antigen"). This is important for infectious diseases with recurrent infections (influenza) or antigenic evolution (HIV, malaria). For influenza, neutralizing antibodies are primarily generated against the viral hemagglutinin (HA) head domain. Different virus strains have different HA head regions that 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.

[0398] The mechanisms of traces of the original antigen in the vaccine may be due to epitope exclusion (pre-existing antibodies, especially mucosal IgA, shield the vaccine from all antigen-presenting cells (APCs); dendritic cell pathways (memory B cells internalize the new vaccine, reducing 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).

[0399] To overcome traces of the original antigen in the vaccine, one can increase the dose (e.g., high-dose vaccines (three times the flu dose for patients over 60 years old)); encapsulation (putting the vaccine into an emulsion or liposome to preferentially deliver the vaccine to dendritic cells (Shingrix, varicella zoster vaccine)); and / or dendritic cell activators (TLR agonists may increase the number and diversity of activated T cells against vaccine antigens).

[0400] To study the original antigen traces in mouse models, the following methods can be used: hapten-protein conjugates (hapten is a small molecule, such as fluorescein or DNP, which can be conjugated to protein antigens such as ovalbumin and KLS); or pre-immunization with unconjugated protein antigen to suppress the antibody immune response to hapten-protein conjugates. For influenza in these models, hyperimmunization is performed against one strain of virus with one protein (e.g., influenza HA), boosted with a partially cross-reactive HA from another strain of virus, and then the B and T cell immune responses to the second HA are analyzed, including the epitopes recognized, clonal diversity by nexgen RNA sequencing, and neutralization capacity, which are then correlated with in vivo protection.

[0401] Shingrix is ​​a recombinant VSV glycoprotein E, nonphospholipid A from Salmonella, and the QS-21 saponin molecule in a liposomal formulation made with dioleoylphosphatidylcholine and cholesterol in buffered saline that is reconstituted at the time of use. To make a flu vaccine similar to Shingrix, the vaccine has a protein antigen, two adjuvants in a liposomal formulation.

[0402] Example 4

[0403] Due to antigenic drift of influenza viruses, the effectiveness of influenza vaccines remains 10-60% each year. Synthetic TLR4 and TLR7 agonists (1Z105 and 1V270) enhance the production of Th2- and Th1-mediated anti-hemagglutinin antibodies, respectively. The combination of 1Z105 and 1V270 promotes balanced Th1 / Th2 immunity and prevents influenza virus infection. To enhance adjuvant efficacy, a structure-activity relationship study of 1Z105 was performed, and a derivative with higher in vitro efficacy, 2B182C, was identified. In in vivo vaccination studies using the model antigen ovalbumin, 2B182C induced higher serum IgG1 levels and additionally enhanced the release of antigen-specific IgG2a induced by 1V270. In addition, liposomal formulations of 2B182C and 1V270 reduced cytotoxicity and reactogenicity, and maintained activity in enhancing Th1- and Th2-mediated antibody production. In in vivo vaccination studies using inactivated A / California / 04 / 2009(H1N1)(pdm09) as antigen, this liposomal combination adjuvant increased the number of T follicular helper cells, germinal center B cells, and antibody-secreting plasma cells. Next-generation sequence analysis of B and T lymphocytes in draining inguinal lymph nodes showed that this combination adjuvant increased B cell clonotypes of immunoglobulin heavy chain (IGH) genes, shared B cell clones, and TCR clonality. These findings suggest that the combination contributes to enhanced antigen-specific Th1 immune responses. Finally, the vaccine with the combination adjuvant protected against lethal homologous virus challenge with less lung damage.

[0404] method

[0405] Mouse

[0406] Female 6- to 8-week-old BALB / c mice were purchased from Jackson Laboratories (Bar Harbor, MA). Animal experiments using ovalbumin or inactivated influenza virus as antigens that did not require live virus challenge were performed at the University of California, San Diego Animal Facility. Influenza challenge studies were performed at the Utah State University Animal Research Center (Charles River Laboratories, Wilmington, MA) using 6-week-old female BALB / c mice. All animal experiments were approved in advance by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego or Utah State University.

[0407] Cells and reagents

[0408] TLR4 / NF-kB reporter cell line HEK-Blue TM humanTLR4 and HEK-Blue TMmurineTLR4 cells were purchased from InvivoGen (Catalog No., San Diego, California). Mouse primary BMDCs were prepared from bone marrow cells harvested from the femur of C57BL / 6 mice. BMDCs were treated with the specified compounds in RPMI supplemented with 10% FBS (Omega, Tarzana, California) and penicillin / streptomycin (100 units / mL / 100 μg / mL, ThermoFisher Scientific, Waltham, Massachusetts). Monophospholipid A (MPLA), AddaVax was purchased from InvivoGen (Catalog No., San Diego, California). Inactivated influenza A virus [A / California / 04 / 2009 (H1N1) pdm09] (IIAV) was obtained from BEI Resources (#NR-49450, Manassas, Virginia). TLR7 agonist 1V270, TLR4 agonist 1Z105 and its derivatives, including 2B182C, were synthesized. Liposomal formulations of 1V270 (20 μM), 2B182C (4 mM), and 1V270 + 2B182C (20 μM + 4 mM) were prepared at Innimune, Inc. (Missoula, MT).

[0409] TLR4 / NF-κB reporter cell assay

[0410] TLR4 / NF-κB activation using HEK-Blue TM hTLR4 and HEK-Blue TM mTLR4 (InvivoGen) was evaluated. Cells were treated with 1Z105 and 2B182C (2-fold serial dilutions starting from 10 μM) for 20 hours. NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) protein was measured in culture supernatants according to the manufacturer's protocol.

[0411] Evaluation of in vivo antibody production

[0412] BALB / c mice were immunized intramuscularly (im) with IAV (10 μg / injection) plus the indicated adjuvants in the gastrocnemius muscle of the hind leg on days 0 and 21. Detailed adjuvant concentrations and number of animals in each treatment are described in each figure legend. Sera were 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 analysis of 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) was used as the vehicle.

[0413] NGS testing of BCR and TCR repertoires

[0414] Immunization regimen Fig.28 As shown in A. Briefly, mice were sacrificed on day 28 and inguinal lymph nodes were harvested. Total RNA was extracted from lymphocytes (bulk) using the 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 library analysis technology (Repertoire Genesis Inc., Osaka, Japan).

[0415] Assessing protection against lethal influenza virus challenge

[0416] BALB / c mice were inoculated with formulated 1V270 and 2B182C and IIAV (3ug / injection) by intramuscular injection on day 0, and were infected intranasally with homologous or heterologous influenza A viruses A / California / 04 / 2009 (pdmH1N1) and A / Victoria / 3 / 75 (H3N2) on day 21. The immunizing dose of IIAV; 3μg / injection was determined in preliminary experiments to protect 30-50% of animals from homologous virus challenge. For influenza virus challenge, mice in the group were anesthetized by intraperitoneal injection of ketamine / xylazine (50mg / kg / / 5mg / kg), and then each mouse was treated with 1×10 5 (3×LD 50 ) cell culture infectious dose (CCID 50 Each mouse was injected with 5 × 10 2 (3×LD 50 )CCID 50Influenza A / Victoria / 3 / 75 (H3N2) virus was challenged intranasally. All mice were challenged with the virus on the 21st day of the study. Influenza virus (H1N1pdm), strain number 175190, from Dr. Elena Govorkova (Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee). The virus was adapted to replicate in the lungs of BALB / c mice by 9 consecutive passages in mice. The virus was plaque purified in Madin-Darby Canine Kidney (MDCK) cells and the virus stock was prepared by growing in chicken embryos and then growing in MDCK cells. Influenza A / Victoria / 3 / 75 (H3N2) virus was obtained from the American Type Culture Collection (Manassas, Virginia). The virus was not lethal to mice at first, but became lethal after 7 consecutive passages in the lungs of infected animals. After adaptation in mice, the virus stock was prepared by growing in MDCK cells.

[0417] Determination of lung virus titers and lung inflammation

[0418] 6 days after virus attack, bronchoalveolar lavage (BAL) procedure was performed immediately after blood sampling, and completed within 5 to 10 minutes after each animal died. 0.75mL of phosphate buffered saline (PBS) was slowly delivered to the lungs through an endotracheal tube. After delivery, liquid was slowly extracted by gentle suction immediately, and the sample was stored at -80 ° C. The program was repeated 3 times in total, and the lavage fluid from each mouse was collected. In order to measure lung virus titer, BAL samples were centrifuged at 2000g for 5 minutes. Different 10-fold dilutions of BAL supernatant were measured three times, the infectious virus in MDCK cells was measured, and the virus titer was calculated. In order to measure lung cytokine levels, according to the instructions of the manufacturer (Quansys Biosciences Q-PlexTM Array, Logan, Utah), the method based on chemiluminescence multiple ELISA analysis was used to test the MCP-1 and IL-6 in the sample (200 μL) from each lung lavage fluid.

[0419] Hemagglutination inhibition titer

[0420] For hemagglutination inhibition (HI) titers, sera were pretreated with receptor destroying enzyme II (RDE; Vibrio cholerae neuraminidase; YCC-340; Accurate Chemical and Scientific, Westbury, NY) in a dilution of three parts enzyme to one part serum and incubated at 37°C for 18 hours to remove nonspecific inhibitors. RDE was then inactivated by heating at 56°C for 45 minutes. Serum samples were diluted with PBS in 96-well round-bottom microtiter plates (Fisher Scientific, Pittsburgh, PA). After serum dilution, 8 HA units / well of influenza A / CA / 04 / 2009 (H1N1pdm) or influenza A / Victoria / 3 / 75 (H3N2) virus + turkey erythrocytes (Lampire Biolabs, Pipesville, PA) were added (50 μL per well), mixed briefly, and incubated at room temperature for 60 minutes. HI titers of serum samples were expressed as the reciprocal of the highest serum dilution that completely inhibited hemagglutination.

[0421] Virus neutralization titer

[0422] For the anti-influenza virus neutralizing antibody assay, MDCK cells were plated at 1 × 10 4 Cells were seeded in MEM medium (minimum essential medium) containing 5% FBS (Hyclone, Logan, Utah) in 96-well plates. Serial 2-fold dilutions of serum samples were prepared in serum-free medium containing 10 units / mL trypsin and 1 μg / mL EDTA, starting from 1:32 dilution and ending with 1:4096 dilution. Each serum dilution was mixed with serum-free medium (containing trypsin and EDTA) containing approximately 100 CCID50 / well H1N1pdm or influenza A / Victoria / 3 / 75 (H3N2) virus at 1:1 (0.1 mL). After culturing for 1 hour at room temperature, the serum-influenza virus mixture (0.2 mL) was transferred to the wells containing MDCK cells and cultured for 3 days. Anti-influenza virus neutralizing antibodies were measured by cytopathic effect (CPE) inhibition. CPE was scored based on duplicate samples by examining the MDCK cell monolayer under an optical microscope on the 3rd day after infection.

[0423] Statistical analysis

[0424] Data obtained in the in vivo study are presented as mean and standard error of the mean (SEM), and in vitro data are presented as mean and standard deviation of the mean (SD). For in vitro data, two-tailed Welch t-test was used to compare the two groups. For antigen-specific antibodies, flow cytometric analysis of immune cell numbers, BCR-seq, TCR-seq, lung virus titers, HI endpoint titers, and VN endpoint titers, Kruskal-Wallis test, and Dunn post hoc test were used. The correlation between lung virus titers and cytokine / chemokine levels was analyzed using Spearman rank correlation test. For body weight, the area under the curve was calculated for each mouse, and statistical analysis was performed using one-way analysis of variance. The 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.

[0425] Table 8. Reagents used in ELISA assays for hIL-8, mIL-12, and mIL-6

[0426]

[0427] Table 9. Reagents used in ELISA assays for hIL-8, mIL-12, and mIL-6

[0428]

[0429]

[0430] Table 10. Reagents used in ELISA assay for IgG

[0431]

[0432]

[0433] result

[0434] The structure-activity relationship study of 1Z105 yielded 2B182C

[0435] In order to improve the potency of the small molecule pyrimidoindole TLR4 ligand 1Z105, a structure-activity relationship analysis (to be filled in by the chemist) was performed. A total of 56 compounds were synthesized and screened using human and mouse HEK TLR4 reporter cells (HEK-BluemTLR4 and hTLR4, respectively). Among these SAR compounds, 2B182C was found to be a derivative with higher TLR4 stimulatory potency in both mouse and human reporter cells. The EC of 2B182C was investigated using HEK TLR4 reporter cells.50 And with 1Z105's EC 50 For comparison ( Fig.21 B) EC with 1Z105 50 In contrast, 2B182C showed EC 50 These data indicate that the SAR study successfully yielded a derivative that exhibited a higher potency for stimulating TLR4, especially human TLR4.

[0436] TLR4 agonist 2B182c enhances antigen-specific IgG1 production

[0437] The TLR4 agonist 1Z105 induces Th2-mediated IgG1 production, and the TLR7 agonist 1V270 enhances Th1 cell immunity to influenza virus (Goff et al., J. Virol. , 89 :3221(2015); Goff et al., J. Virol. , 91 :e01050(2017)). It was hypothesized that the efficacy of the TLR4 agonist 2B182C as an influenza vaccine adjuvant could be improved by combining it with 1V270. Therefore, it was investigated whether the combination of 2B182C and 1V270 improved the efficacy of the adjuvant in vivo compared to the combined adjuvant of 1Z105+1V270.

[0438] To develop an effective combination vaccine adjuvant, the efficacy of 1Z105 and 2B182C and the optimal dose as a single agent were compared using inactivated influenza A virus [A / California / 04 / 2009(H1N1)pdm09] (IIAV) as an antigen. BALB / c mice were immunized with IIAV mixed with TLR4 agonist 1Z105 or 2B182C on days 0 and 21, and blood was collected on day 28 ( Fig. 22 A). Antibodies (IgM, IgG1, and IgG2a) against two viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), were evaluated in serum by ELISA. 1Z105 and 2B182C were dissolved in DMSO to a final concentration of 10%. The results showed that the higher dose of 2B182C (200 nmol / injection) significantly increased the IgG1 antibodies against HA and NA ( Fig. 22 B). Interestingly, 2B182C, but not 1Z105, enhanced anti-NA-specific IgG2a ( Fig. 22 C). 2B182C only slightly increased anti-HA IgM levels ( Fig.24 A).

[0439] The combination of 2B182C and the TLR7 agonist 1V270 increases antigen-specific IgG1 and IgG2a

[0440] Next, we analyzed the co-adjuvant effect of these TLR4 agonists on antibody production when combined with the 1 nmol / injection dose of the TLR7 agonist 1V270, which has been reported to induce IgG2a production and enhance Th1 immune responses (Goff et al., 2017). The results showed that while 1V270 alone only induced anti-HA IgG2a production, when it was combined with 2B182C, both anti-HA and anti-NA IgG1 and IgG2a antibodies were significantly induced. This suggests that these compounds may act in an additive manner ( Fig.23 A and 23B). On the other hand, 1Z105 failed to enhance 1V270-induced IgG2a production. Animals in the 1V270+2B182C-group produced higher amounts of IgG1 and IgG2a, and the immune balance was biased toward Th1-mediated IgG2a production, indicating that this treatment helped enhance Th1 immune responses ( Fig.23 C). The combination of 1V270 and 2B182C showed a moderate effect on anti-HA IgM production ( Fig.24 B).

[0441] In summary, the combination of 200 nmol / injection 2B182C + 1 nmol / injection 1V270 induced the highest amounts of antigen-specific IgG1 and IgG2a, as well as Th1-biased immune responses, which are desirable for protection in influenza virus infection. Therefore, this combination was selected for the next formulation.

[0442] Liposomal formulation of 2B182C reduces cytotoxicity

[0443] In view of the above results, a 1V270 / 2B182C ratio of 1 / 200 (TLR4 / TLR7) was used ([1 nmol / injection (20 μM) 1V270 and 200 nmol / injection (4 mM) 2B182C]. In the development of vaccine adjuvants, it may be important to adjust the formulation of the compounds in order to avoid unwanted cytotoxicity and reactogenicity while maintaining the response to the vaccine. Therefore, 1V270 and 2B182c were formulated in liposomes by Inimmune (Missoula, MT). The activity of the formulated compounds was tested in primary mouse BMDCs. These formulated compounds maintained IL-12 secretion levels similar to those of the DMSO formulated compounds ( Fig.25A ). Liposome formulation significantly improved the cytotoxicity induced by DMSO-2B182C or DMSO-1V270+2B182C. ( Fig.25B Histological analysis by H&E staining of the injected muscle was performed as shown in Fig.25C Multiplex cytokine / chemokine analysis of serum after compound administration is shown in Fig.25D shown.

[0444] Liposomes 1V270 and 2B182C synergistically enhance the production of anti-HA and anti-NA IgG1 and IgG2a

[0445] use Fig. 22 The in vivo adjuvant properties of the compounds were evaluated using the prime-boost protocol described in A. Antigen-specific antibodies were assessed in sera harvested on day 28 by ELISA. The results showed that lipo-2B182C induced higher levels of IgG1, which was consistent with DMSO-2B182C ( Fig.26 A). Unlike DMSO-1V270, lipo-1V270 alone did not promote the production of IgG2a ( Fig.26 B). Although each agonist had a small effect on IgG2a, when the two adjuvants were combined, the production of antigen-specific antibodies was synergistically enhanced ( Fig.26 B). On the other hand, the total IgG levels induced by liposome vectors 1V270, 2B182C and 1V270+2B182C were comparable ( Fig.26 C). Antigen-specific IgM levels were not affected by any treatment ( Fig. 27 Consistent with the trend observed with the DMSO formulation, the liposome combination adjuvant produced an immune balance that favored Th1 ( Fig.26 D).

[0446] Formulated 1V270+2B182C enhances antibody secretion response

[0447] In order to study whether the formulated adjuvant induces B cell activation that promotes antigen-specific antibody secretion, flow cytometry was used to investigate the Tfh cells, GC B cells, plasmablasts, and plasma cells of lymphocytes in the inguinal lymph nodes. Using the above-mentioned immunization protocol, lymphocytes in the inguinal lymph nodes were harvested on day 28 and analyzed by flow cytometry ( Fig.28 A and 28B). As a result, the percentage of Tfh cells identified as CD3+CD4+PD-1+CXCR5+ cells was greatly increased by using lipo-1V270+2B182C ( Fig.28 B and Fig.29 ). In addition, the combined adjuvant increased the percentage of GC B cells (CD3-CD19+CD95+GL7+). Increases in plasmablasts and plasma cells were observed in mice immunized with lipo-1V270+2B182C. The results show that the combined adjuvant enhances GC responses compared to single agents.

[0448] 1V270 and 2B182C combined adjuvants increase BCR diversity and TCR clonality

[0449] To investigate whether the combined adjuvants affect the diversity of BCR, next generation sequencing analysis of the IGH gene was performed (performed by Repertoire Genesis, Osaka, Japan). A prime-boost IIAV model was used, and lymphocytes from the inguinal lymph nodes were collected on day 28 ( Fig.30 A). BCR sequence analysis showed that the BCR diversity normalized to the total read length (Total reads) was significantly increased by lipo-1V270+2B182C ( Fig.30 A). The clonotypes of the IgG gene were analyzed by similarity analysis, which compared the IGH clones of two mice in the same group to see if there were shared clones and calculated the Jacquard index; Jacquard index: J(A, B) = (A∩B) / (A∪B) ( Fig.30 B). lipo-1V270+2B182C significantly increased the Jacquard index of IGH, IGHG1, and IGHG2A, indicating an increase in clones shared by two mice within the group. In addition, in the lipo-1V270+2B182c group, 3 mice had a total of 6 clones (0.03%). These results indicate that the liposome combination adjuvant increased the BCR diversity of total IGH and IGHG2A. This is consistent with the higher IgG2a levels after immunization with the combination adjuvant. The shared clones detected in the combination adjuvant immunization group may recognize dominant epitopes of the antigen. TCR sequencing was performed to examine whether the formulation of the adjuvant helps to increase the f TCR clonality against the antigen. It is expected that the combination adjuvant and lipo-2B182C increase the clonality of TCRα and TCRβ ( Fig.30 C). Overall, animals immunized with lipo-1V270+2B182C showed higher BCR diversity and TCR clonality. This may support the data that the combined adjuvant enhances Th1 responses.

[0450] Lipo-2B182C and lipo-1V270+2B182C protected mice from homologous influenza virus.

[0451] The combined adjuvant induces a Th1-biased immune response, with a high clonality of diverse BCRs and TCRs. To test whether this diversity could indicate an immune response against 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 (single dose) homologous (H1N1) influenza virus on day 21 post-inoculation. The body weight and survival of the mice were monitored for an additional 21 days ( Fig.31 A). Lipo-2B182C and lipo-1V270+2B182C significantly inhibited weight loss after viral infection ( Fig.31B). In addition, lipo-1V270 showed 90% protection, and lipo-2B182C and lipo-1V270+2B182C completely protected mice from homologous influenza virus ( Fig.31 C). To evaluate whether the survival of mice is associated with the viral titer in the lung, bronchoalveolar lavage was performed to measure the viral titer in the lavage fluid. The results showed that lipo-1V270+2B182C effectively suppressed the viral titer in the lung on day 6 ( Fig.31 D). In humans, there is upregulation of cytokines and chemokines in airway epithelial cells (e.g., MCP-1, IL-6, etc.) associated 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 pro-inflammatory cytokines (IL-6) and chemokines (MCP-1) in lung fluid using Quansys multiplex ELISA. The results showed that the combined liposomal adjuvant significantly inhibited the production of MCP-1 and IL-6 ( Fig.31 E). The levels of proinflammatory cytokines were correlated with lung virus titers [MCP-1 (P<0.0001, Spearman correlation coefficient r=0.83), IL-6 (P<0.0001, Spearman correlation coefficient r=0.79) ( Fig.31 F). This trend was further enhanced in the lipo-1V270+2B182C group. These results suggest that the combined adjuvant reduces lung damage by inhibiting viral entry and proliferation after infection. To evaluate whether protection is associated with hemagglutination inhibition titer (HI) and virus neutralization titer (VN), sera were collected on day 21 after immunization and examined for HI and VN ( Fig.31 A). Compared with the unimmunized group, increased HI titers were observed in 19 out of 20 mice in the lip-1V270, lipo-2B182C, and lipo-1V270+2B182C groups ( Fig.31 G). In addition, lipo-2B182c and lipo-1V270+2B182C induced significantly higher VN ( Fig.31 H). VN titer was negatively correlated with lung virus titer (P<0.01, Spearman correlation coefficient r=-0.59, Fig.31 I). Protection against heterologous influenza virus A / Victoria 3 / 75 (H3N2) was assessed using the same protocol as the homologous challenge experiment ( Fig.31 A). There were no significant differences in weight loss, survival, and lung virus titers compared with the liposome control group ( Fig.32 AC). Overall, the formulated combination adjuvant showed significant protection against the homologous H1N1 virus without adverse inflammatory effects, although it was not sufficient to provide heterologous protection.

[0452] Table 11. Number of shared clones of total IgG genes in BCR-seq

[0453]

[0454]

[0455] Example 5

[0456] Liposome co-encapsulation of 1V270 (TLR7 ligand) and 2B182C (TLR4 ligand) broadens the antibody epitope

[0457] Universal vaccines for influenza virus infection need to induce antibodies that recognize a wide range of epitopes of major antigenic molecules, hemagglutinin (HA) and neuraminidase (NA). Therefore, the epitope expansion and cross-reactivity of antibodies induced by combined adjuvants (1V270 and 2B182C) were investigated. BALB / c mice (n=5-10) were immunized with inactivated virus and liposome preparations 1V270 (Lipo-1V270), 2B182C (Lipo-2B182C), co-encapsulated liposomes 1V270+2B182C [Lipo-(1V270+2B182C)], and liposomes Lipo-1V270 and liposomes Lipo-2B182C alone. Blank liposomes were used as controls, immunized on day 0 (primary immunization) and day 21 (boost immunization), and serum was collected on day 28.

[0458] Epitope expansion was assessed by HA peptide ELISA. The end-to-end HA peptide matrix (139 peptides) of influenza A (H1N1) pdm09 virus was obtained from BEI Resources. Pooled peptides containing 5 consecutive peptides (28 peptide pools in total) were inoculated onto the ELISA plate. The reactivity of 1:200 diluted sera to each peptide pool was tested by OD405-570. The OD of each serum was plotted on a heat map ( Fig.38 A) and the mean OD of individual animals were compared. The sera of mice vaccinated with co-encapsulated liposomes 1V270+2B182C [Lipo-(1V270+2B182C)] showed significantly higher OD ( Fig.38 B). These data indicate that Lipo-(1V270+2B182C) induces antibody responses that recognize multiple HA epitopes.

[0459] To test whether the recognition of a broad range of HA epitopes induced by Lipo-(1V270+2B182C) is associated with cross-protection against influenza virus infection with different subtypes, we tested the cross-reactivity of the antibodies against various subtypes of HA and NA by ELISA ( Fig.39 and 40 ). Subtypes HA and NA belonging to different phylogenetic distances. The IgG geometric mean titer (GMT) of mice immunized with co-encapsulated Lipo-(1V270+2B182C) showed high reactivity not only with HA in group 1 (H1, H11, H12), but also with HA in group 2 (H3 and H7). The reactivity was also observed to be broadened to different subtypes of NA. In summary, the antibodies produced by animals vaccinated with IIAV+Lipo-(1V270+2B182C) are highly cross-reactive to HA and NA of different subtypes.

[0460] All publications, patents and patent applications are incorporated herein by reference. Although the present invention has been described in the foregoing specification with respect to certain preferred embodiments of the present invention, and many details have been set forth for illustrative purposes, it will be apparent to those skilled in the art that the present invention is susceptible to other embodiments, and that certain details described herein may vary greatly without departing from the basic principles of the present invention.

Claims

1. A pharmaceutical preparation comprising liposomes, wherein the liposomes contain a TLR4 agonist and a TLR7 agonist, Wherein the TLR7 agonist is: And wherein the TLR4 agonist is: ， The liposomes include (i) at least one of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphorylcholine (PC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and (ii) cholesterol.

2. The formulation according to claim 1, wherein the TLR4 agonist and the TLR7 agonist are administered simultaneously.

3. The formulation of claim 1 or 2, further comprising one or more immunogens.

4. The formulation of claim 3, wherein the immunogen is a microbial immunogen.

5. The preparation according to claim 4, wherein the microorganism is a virus or a bacterium. The preparation according to claim 1 or 2, wherein the content of the TLR7 agonist is 0.01 to 100 nmol.

7. The preparation according to claim 1 or 2, wherein the content of the TLR7 agonist is 0.1 to 10 nmol.

8. The preparation according to claim 1 or 2, wherein the content of the TLR7 agonist is 100 nmol to 1000 nmol.

9. The preparation according to claim 1 or 2, wherein the content of the TLR4 agonist is 2 nmol to 20 μmol. 10 . The preparation according to claim 1 or 2 , wherein the content of the TLR4 agonist is 20 nmol to 2 μmol. The preparation according to claim 1 or 2, wherein the content of the TLR4 agonist is 2 μmol to 100 μmol.

12. The formulation according to claim 1 or 2, wherein the ratio of TLR7 and TLR4 agonists is 1:

10.

13. The formulation according to claim 1 or 2, wherein the ratio of TLR7 and TLR4 agonists is 1:

100.

14. The formulation according to claim 1 or 2, wherein the ratio of TLR7 and TLR4 agonists is 1:

200.

15. The formulation according to claim 1 or 2, wherein the ratio of TLR7 and TLR4 agonists is 5:

20.

16. The formulation of claim 1 or 2, wherein the ratio of TLR7 and TLR4 agonists is 5:

100.

17. The formulation of claim 1 or 2, wherein the ratio of TLR7 and TLR4 agonists is 5:200.

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