Immunostimulatory compositions and use methods thereof

Abstract

To provide a lipid conjugate and a method for enhancing cargo delivery to the lymph nodes.SOLUTION: The lipid conjugate typically includes three domains: a lipophilic domain that binds to albumin, a polar block domain, and a cargo such as a molecular adjuvant or immunostimulatory compound (such as an oligonucleotide) or antigenic peptide. Depending on the cargo, the length and compositions of the polar block can be tailored to push the equilibrium toward albumin binding, stable micelle formation, or cell insertion. The conjugate can be administered to a subject, for example, a subject with cancer or an infection, to induce or enhance a robust immune response in the subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED INVETIONS This application claims the benefit of U.S. Provisional Patent Application No. 61 / 620,518, filed April 5, 2012, the entirety of which is incorporated herein by reference.

[0002] The present invention relates to the field of vaccine technology, and more specifically to albumin-binding lipids that are conjugated to cargoes and efficiently target said cargoes to lymph nodes. [Background technology]

[0003] BACKGROUND OF THEINVENTION Subunit vaccines present antigens to the immune system without introducing viral particles in an attempt to generate an effective immune response against the antigen. Such subunit vaccines are often poorly immunogenic and require co-administration of one or more adjuvants to generate an effective immune response (Perrie, Y., et al., Int. J. Pharm. 364, 272-280 (2008); Zepp, F. Vaccine 28S C14-C24 (2010)). Immunostimulatory oligonucleotides (e.g., those containing unmethylated cytosine-phosphate-guanine ("CG" or "CpG") motifs) can be used as adjuvants to stimulate both cellular and humoral immune responses (Vollmer, J. & Krieg, AM Adv. Drug Delivery Rev. 61, 195-204 (2009); Klinman, DM Nat. Rev. Immunol. 4, 249-259 (2004)). The difficulty in the clinical application of oligonucleotides as vaccine adjuvants is the lack of an efficient system for targeting the oligonucleotides to immune cells of the lymphatic system in vivo (Von Beust, BR, et al. Eur. J. Immunol. 35, 1869-1876 (2005); Bourquin, C., et al., J. Immunol. 181, 2990-2998 (2008)).

[0004] Transporting antigens / adjuvants from the injection site to secondary lymph nodes is difficult and depends on the complex physiology of the lymphatic system (Pal, I. & Ramsey, JD Adv. Drug Delivery Rev. 63, 909-922 (2011); Reddy, ST, et al., Nat. Biotechnol. 25, 1159-1164 (2007)). Antigens / adjuvants introduced into the body may be taken up by immune dendritic cells (DCs) at the injection site and then carried via DC trafficking (e.g., cells associated with antigens or large particles (>200 nm)) to lymph nodes. Alternatively, they may enter lymphatic vessels directly and be excreted in secondary lymphoid organs (e.g., small particles (<200 nm)) where a significant portion of immune cells reside (Bachmann, MF & Jennings, GT Nat. Rev. Immunol. 10, 787-796 (2010); Reddy, ST, et al., Nat. Biotechnol. 25, 1159-1164 (2007); Singh, M. Vaccine adjuvant and delivery system. Wiley. (2007); Oyewumi, MO, et al., Expert Rev. Vaccines 9, 1095-1107 (2010); Cai, S., et al., Adv. Drug Delivery Rev. 63, 901-908 (2011); Manolova, V., et al. Eru. J. Immunol. 38, 1404-1413 (2008)).

[0005] Soluble antigen / adjuvant compounds drain through lymph nodes within hours, providing only a short exposure to the vaccine (Pape, et al., Immunity 26, 491-502 (2007)). Attempts to enhance delivery of antigens / adjuvants to lymph nodes after parenteral injection have included the use of depot-forming adjuvants or particulate carriers that are preferentially internalized by antigen-presenting cells (Johansena, et al., Journal of Controlled Release, 148, 56-62 (2010), Moon, et al., Adv. Mater., 24, 3724-3746 (2012), Bachmann and Jennings, Nat. Rev. Immunol. 10, 787-796 (2010), Hubbel, et al., Nature, 462, 449-460 (2009), Pal, & Ramsey, JD Adv. Drug Delivery Rev., 63, 909-922 (2011), Reddy, et al., JA Nat. Biotechnol., 25, 1159-1164 (2012)). (2007), John, et al., Nature Materials, 11, 250-257 (2012)), but these approaches did not reach the efficacy of direct injection of vaccines into lymphoid tissues (Senti, et al., Curr. Opin. Allergy Clin. Immunol., 9:537-543 (2009)). Molecularly targeted vaccines based on conjugation of antigens to antibodies or other ligands targeting dendritic cells not only deliver DCs to draining lymph nodes, but also drain into the systemic circulation, allowing DCs to reach distant tissues (Keler, et al., Oncogene, 26, 3758-67 (2007), Tacken, et al., Nat. Rev. Immunol., 10, 790-802 (2007), Tenbusch, et al., PLoS ONE, 7, e39038 (2012)).Such systemic delivery may promote tolerance unless inflammatory adjuvants are also co-administered systemically, an approach that may result in unacceptable toxicity in a prophylactic vaccine.

[0006] However, there is still a need for an effective delivery system to target antigens / adjuvants to lymphatic antigen-presenting cells, particularly CD8+ DCs, a step important for inducing cytotoxic T lymphocyte (CTL) responses, since CD8+ DCs are the primary DCs capable of cross-presentation, a process required for presenting extracellular antigens in MHC class I molecules to CD8+ T cells (Smith, CM, et al., J. Immunol. 170, 4437-4440 (2003); Schnorrer, P., et al., Proc. Natl. Acad. Sci. USA 103, 10729-10734 (2006); Bedoui, S., et al., Nat. Immunol. 10, 488-495 (2009)).

[0007] It is therefore an object of the present invention to provide compositions and methods for increasing the delivery of vaccine adjuvants to lymph nodes.

[0008] It is also an object of the present invention to provide compositions and methods for increasing the delivery of vaccine antigens to lymph nodes.

[0009] Another object of the present invention is to provide immunogenic compositions and methods of use thereof to increase the delivery of vaccine adjuvant and antigen combinations to lymph nodes.

[0010] It is a further object of the present invention to provide immunogenic compositions and methods of use thereof for inducing an immune response.

[0011] Another object of the present invention is to provide compositions and methods for increasing retention of vaccine adjuvants and antigens locally at the site of administration and at the ipsilateral draining lymph nodes.

[0012] It is a further object of the present invention to provide a method for increasing a local immune response. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Perrie, Y., et al., Int. J. Pharm. 364, 272-280 (2008) [Non-Patent Document 2] Zepp, F. Vaccine 28S C14-C24 (2010) [Non-Patent Document 3] Vollmer, J. & Krieg, AM Adv. Drug Delivery Rev. 61, 195-204 (2009) [Non-Patent Document 4] Klinman, DM Nat. Rev. Immunol. 4, 249-259 (2004) [Non-Patent Document 5] Von Beust, BR, et al. Eur. J. Immunol. 35, 1869-1876 (2005) [Non-Patent Document 6] Bourquin, C., et al., J. Immunol. 181, 2990-2998 (2008) [Non-Patent Document 7] Pal, I. & Ramsey, JD Adv. Drug Delivery Rev. 63, 909-922 (2011) [Non-Patent Document 8] Reddy, ST, et al., Nat. Biotechnol. 25, 1159-1164 (2007)

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Summary of the Invention

Means for Solving the Problems

[0014] Summary of the Invention It has been discovered that albumin-binding lipids can be conjugated to cargo and can effectively target the cargo to lymph nodes in vivo. Upon in vivo introduction, the lipid conjugates are believed to bind to endogenous albumin, preventing the conjugates from rushing into the bloodstream, and instead retargeting them to the lymphatic system and draining lymph nodes, where they accumulate due to filtering of albumin by antigen-presenting cells. When the lipid conjugates contain immune stimulants (e.g., immune stimulating oligonucleotides or antigenic peptides), the conjugates can induce or enhance strong immune responses. The amphiphilic albumin binding conjugates are as follows: (a) lipid components; (b) any polar component; and (c) an immunomodulatory compound or molecular adjuvant; Including, wherein the immunomodulatory compound or molecular adjuvant is conjugated directly to the lipid or to the lipid via a linker, wherein the conjugate is sufficiently soluble such that the lipid binds to albumin under physiological conditions, and wherein a plurality of the conjugates can spontaneously form micelles in aqueous solution.

[0015] Lipid conjugates, including lipid-oligonucleotide conjugates and lipid-peptide conjugates, and their use for stimulating immune responses are disclosed. For example, amphipathic oligonucleotide conjugates for targeting lymph nodes can include immunostimulatory oligonucleotides that are (i) directly conjugated to lipids or (ii) linked to linkers that are conjugated to lipids. Typically, the lipids bind to albumin under physiological conditions. In some embodiments, a plurality of the oligonucleotide conjugates can spontaneously form micelles in aqueous solutions that can be disrupted by the addition of an albumin-containing agent. In a specific embodiment, 64% or more of the micelles are disrupted in the presence of 20% fetal bovine serum.

[0016] In some embodiments for targeting lymph nodes, the oligonucleotide comprises an oligonucleotide linker that includes 0, 1, or 2 consecutive guanines. For example, the conjugate has the structure L-5'-G n -ON-3', where "L" is a lipid, "G" is guanine, "n" is 0 to 2, and "ON" is an immunostimulatory oligonucleotide.

[0017] The lipid of the conjugate typically binds to albumin. Exemplary lipids are diacyl lipids, such as diacyl lipids whose chains contain C12 or more hydrocarbon units.

[0018] The immunostimulatory oligonucleotides may be ligands for pattern recognition receptors (e.g., CpG) and may have modified backbones (e.g., phosphorothioate (PS) backbones). In some embodiments, the oligonucleotides comprise 20 or more nucleic acids.

[0019] Conjugates for retention at or near the site of administration are also disclosed. Referred to as micelle-stabilized conjugates, the cargo and the lipid are typically linked by an oligonucleotide linker that includes at least three consecutive guanines. Typically, the conjugates spontaneously form micelles in aqueous solution that are resistant to disruption by albumin. In certain embodiments, more than 36% of the micelles are intact in the presence of 20% fetal bovine serum. In some embodiments, the oligonucleotide conjugates have the structure L-5'-G n -ON-3', where "L" is a lipid, "G" is guanine, "n" is 3 to 10, and "ON" is an immunostimulatory oligonucleotide.

[0020] Lipid-peptide conjugates are also disclosed. Typically, the conjugates include a peptide antigen that is (i) directly conjugated to a lipid or (ii) linked to a linker that is conjugated to a lipid. The lipid typically binds to albumin under physiological conditions. In some embodiments, the peptide antigen, the linker, or a combination of peptide antigen and linker are sufficiently polar to reduce or inhibit the lipid from entering the plasma membrane of a cell.

[0021] Also disclosed is an immunogenic composition comprising lipid-oligonucleotide conjugates, lipid-peptide conjugates, and combinations thereof.The immunogenic composition can be used to increase immune response in a subject.Typically, the subject is administered an effective amount of the immunogenic composition to increase effector immune cell response, for example, to increase the number of CD8+ T cells expressing TNF-α or INF-γ compared to a control.The method can be used to treat a subject with cancer or infectious disease. [Brief description of the drawings]

[0022] [Figure 1-1]FIG. 1A is a schematic diagram illustrating the three domains of a lipid conjugate: a cargo conjugated to a solubility-promoting polar block conjugated to a lipophilic tail. FIG. 1B is a schematic diagram illustrating an exemplary lipid-oligonucleotide conjugate comprising an immunostimulatory oligonucleotide (CpG) cargo conjugated to a lipophilic tail. FIG. 1C is a schematic diagram illustrating an exemplary lipid-peptide conjugate comprising an antigenic peptide cargo conjugated to a polar block conjugated to a lipophilic tail. FIG. 1D is an exemplary lipid-oligonucleotide conjugate comprising a diacyl lipid tail conjugated to an oligoguanine linker conjugated to an oligonucleotide cargo. FIG. 1E is an exemplary lipid-peptide conjugate comprising a diacyl lipid tail conjugated to a polyethylene glycol (PEG) linker conjugated to a peptide cargo. Figure IF is a series of plots showing fluorescence resonance energy transfer (FRET) of fluorescein-labeled free CpG alone (left), fluorescein-labeled free CpG mixed with rhodamine-labeled bovine serum albumin (BSA) (center), and rhodamine label alone (right). Figure IG is a series of plots showing fluorescence resonance energy transfer (FRET) of fluorescein-labeled lipo-CpG alone (left), fluorescein-labeled lipo-CpG mixed with rhodamine-labeled bovine serum albumin (BSA) (center), and rhodamine label alone (right).

[0023] [Figure 1-2]FIG. 1A is a schematic diagram illustrating the three domains of a lipid conjugate: a cargo conjugated to a solubility-promoting polar block conjugated to a lipophilic tail. FIG. 1B is a schematic diagram illustrating an exemplary lipid-oligonucleotide conjugate comprising an immunostimulatory oligonucleotide (CpG) cargo conjugated to a lipophilic tail. FIG. 1C is a schematic diagram illustrating an exemplary lipid-peptide conjugate comprising an antigenic peptide cargo conjugated to a polar block conjugated to a lipophilic tail. FIG. 1D is an exemplary lipid-oligonucleotide conjugate comprising a diacyl lipid tail conjugated to an oligoguanine linker conjugated to an oligonucleotide cargo. FIG. 1E is an exemplary lipid-peptide conjugate comprising a diacyl lipid tail conjugated to a polyethylene glycol (PEG) linker conjugated to a peptide cargo. Figure IF is a series of plots showing fluorescence resonance energy transfer (FRET) of fluorescein-labeled free CpG alone (left), fluorescein-labeled free CpG mixed with rhodamine-labeled bovine serum albumin (BSA) (center), and rhodamine label alone (right). Figure IG is a series of plots showing fluorescence resonance energy transfer (FRET) of fluorescein-labeled lipo-CpG alone (left), fluorescein-labeled lipo-CpG mixed with rhodamine-labeled bovine serum albumin (BSA) (center), and rhodamine label alone (right).

[0024] [Figure 2-1]Figure 2A is a schematic diagram showing the design of an exemplary lymph node targeting amphiphile: a hydrophobic lipid-like tail (L) is conjugated to the 5' end of the CpG ODN, and the CpG sequence has a fully phosphorothioated backbone. Three alternative lipids are shown: cholesterol, acyl (C18), and diacyl (C18). Figure 2B is a line graph showing the results of size-exclusion HPLC of fluorescein-labeled lipid-conjugated CpG after incubation with fetal bovine serum (FBS) for 2 hours at 37°C. Figure 2C is two bar graphs showing in vivo LN (in the left graph, the inguinal lymph node, and in the right graph, the axillary lymph node) accumulation of CpG in various fluorescein-labeled formulations (CpG-F, C18-CpG-F, Cho-CpG-F, lipo-CpG-F, CpG-F in IFA, CpG-F in liposomes) 24 hours after subcutaneous injection of 3.3 nmol fluorescein-labeled CpG. Figure 2D is a line graph showing the kinetics of CpG fluorescence (normalized to injected dose) in LNs after injection of CpG-F (inguinal lymph node (●) and auxiliary lymph node (■)) or lipo-CpG-F (inguinal lymph node (▲) and axillary lymph node (▼)). FIG. 2E is a schematic diagram showing a generalized design of lymph node targeting amphiphiles that include an albumin binding domain, a polar spacer, and a cargo linked to the end of the spacer. FIG. 2F shows that the length of the polar block controls the balance of a three-way equilibrium: intact micelles, albumin-bound amhiphiles, and amhiphiles that have entered the cell membrane. FIG. 2G and FIG. 2H show the effect of varying the length of the poly(ethylene glycol) (PEG) linker of lipo-(PEG)n-FITC conjugates on cell membrane entry and lymph node targeting, where n is the number of 4-unit oligoethylene glycol repeats in the PEG block. FIG. 2G is a bar graph showing quantification of cellular entry of amphiphiles with various PEG lengths. FIG. 2H is two bar graphs showing in vivo LN (inguinal lymph nodes in the left graph and axillary lymph nodes in the right graph) accumulation of amphiphiles in various fluorescein-labeled formulations (Lipo-(PEG)nF (n=1, 2, 4, 6, 8)) 24 hours after subcutaneous injection.Figure 2I is a line graph showing LN uptake of amphiphilic fluorescein-labeled PEG2000 as a function of lipid molecular weight (i.e., length) and Figure 2J is a line graph showing LN uptake of lipid-oligonucleotide conjugates as a function of oligonucleotide length.

[0025] [Figure 2-2]Figure 2A is a schematic diagram showing the design of an exemplary lymph node targeting amphiphile: a hydrophobic lipid-like tail (L) is conjugated to the 5' end of the CpG ODN, and the CpG sequence has a fully phosphorothioated backbone. Three alternative lipids are shown: cholesterol, acyl (C18), and diacyl (C18). Figure 2B is a line graph showing the results of size-exclusion HPLC of fluorescein-labeled lipid-conjugated CpG after incubation with fetal bovine serum (FBS) for 2 hours at 37°C. Figure 2C is two bar graphs showing in vivo LN (in the left graph, the inguinal lymph node, and in the right graph, the axillary lymph node) accumulation of CpG in various fluorescein-labeled formulations (CpG-F, C18-CpG-F, Cho-CpG-F, lipo-CpG-F, CpG-F in IFA, CpG-F in liposomes) 24 hours after subcutaneous injection of 3.3 nmol fluorescein-labeled CpG. Figure 2D is a line graph showing the kinetics of CpG fluorescence (normalized to injected dose) in LNs after injection of CpG-F (inguinal lymph node (●) and auxiliary lymph node (■)) or lipo-CpG-F (inguinal lymph node (▲) and axillary lymph node (▼)). FIG. 2E is a schematic diagram showing a generalized design of lymph node targeting amphiphiles that include an albumin binding domain, a polar spacer, and a cargo linked to the end of the spacer. FIG. 2F shows that the length of the polar block controls the balance of a three-way equilibrium: intact micelles, albumin-bound amhiphiles, and amhiphiles that have entered the cell membrane. FIG. 2G and FIG. 2H show the effect of varying the length of the poly(ethylene glycol) (PEG) linker of lipo-(PEG)n-FITC conjugates on cell membrane entry and lymph node targeting, where n is the number of 4-unit oligoethylene glycol repeats in the PEG block. FIG. 2G is a bar graph showing quantification of cellular entry of amphiphiles with various PEG lengths. FIG. 2H is two bar graphs showing in vivo LN (inguinal lymph nodes in the left graph and axillary lymph nodes in the right graph) accumulation of amphiphiles in various fluorescein-labeled formulations (Lipo-(PEG)nF (n=1, 2, 4, 6, 8)) 24 hours after subcutaneous injection.Figure 2I is a line graph showing LN uptake of amphiphilic fluorescein-labeled PEG2000 as a function of lipid molecular weight (i.e., length) and Figure 2J is a line graph showing LN uptake of lipid-oligonucleotide conjugates as a function of oligonucleotide length.

[0026] [Diagram 3] FIG. 3A is a schematic diagram showing the generalized construction and characterization of G-quadruplex stabilized CpG adjuvants. G-quadruplex stabilized CpG micelles are self-assembled from three segments: immunostimulatory CpG-ODN, an ODN composed of n=1-10 G-quartet forming guanines, followed by a central repeat block containing 10-n non-interacting thymidines, and a diacyl lipid tail. In buffer, the ODN self-assembles into three-dimensional spherical micelles with a CpG corona and lipid core. In the presence of K+, the guanine repeats form a G-quadruplex structure through Hoogsteen hydrogen bonds, stabilizing the micelle structure. The stability of the ODN micelles in the presence of albumin can be programmed by simply varying the number of guanines. Albumin binds to the lipid portion of destabilized micelles (n≦2), in contrast to stabilized micelles (n>2), which limit albumin binding and retain micelle assembly. Figure 3B is a schematic (top) and bar graph (bottom) showing the pyrene excimer fluorescent constructs used to assay the stability of G-quadruplex micelles in the presence of albumin. Figure 3C is a line graph showing the stability profile of G-quadruplex CpG micelles as measured by size-exclusion chromatography in the presence of fetal bovine serum (FBS). Figure 3D is a bar graph showing the percentage of B220+ cells, F4 / 80+ cells, and CD11c+ cells that were CpG positive as determined by flow cytometry. ***, p<0.001; **, p<0.01; *, p<0.05.

[0027] [Figure 4]FIG. 4A is a bar graph showing the percentage of peripheral blood lymphocytes isolated from C57Bl / 6 mice that are H-2Kb / SIINFEKL tetramer positive by flow cytometry 6 days after completion of an immunization protocol involving sc injections on days 0 and 14 with a combination of 10 μg OVA and 1.24 nmol CpG formulations as indicated. FIG. 4B is a bar graph showing quantification of INF-γ and TNF-α positive CD8 T cells after 6 hours of antigen-specific restimulation. FIG. 4C is a line graph showing the correlation between LN CpG fluorescence and immune responses measured by SIINFEKL tetramer staining. FIG. 4D is a bar graph showing spleen weights (mg / g body weight) of CpG, Lipo-G2-CpG, and PBS as an index of relative systemic toxicity of the various treatments. FIG. 4E is a schematic showing the assay design. FIG. 4F is a dot plot showing the effect of LN targeting on immune responses (anti-OVA serum IgG titers 20 days after immunization with various antigen / adjuvant combinations as indicated).

[0028] [Diagram 5]FIG. 5A is a bar graph showing the percentage of CD8+ cells isolated from C57Bl / 6 mice that were HPV-16 E749-57 positive by flow cytometry 6 days after completing an immunization protocol including sc injections on days 0 and 14 of the HPV-16 E7 minimal peptide (E749-57) and in combination with 1.24 nmol CpG as indicated. FIG. 5B is a bar graph showing quantification of INF-γ and TNF-α positive CD8 T cells after 6 hours of antigen-specific restimulation as a measure of the extent of antigen-specific CD8+ T cell responses for minimal peptides (A11, Trp2, and HPV-16 E7). FIG. 5C is a bar graph showing that directing lipid conjugates to peptides (lipopeptides) does not elicit a strong antigen-specific immune response as measured by the frequency of INF-γ and TNF-α positive CD8 T cells after restimulation. Figure 5D is a bar graph showing the efficacy of the amphiphilic vaccine as assayed by an in vivo cytotoxicity experiment 7 days after the final immunization with the Trp2 peptide vaccine. Figure 5E is a Kaplan-Meier curve, and Figure 5F is a line graph showing the tumor area over time in mice treated with subcutaneous (sc) TC-1 tumors treated with the amphiphilic HPV-16 E7 peptide vaccine, soluble vaccine, or no vaccine on days 6, 13, and 19 after challenge with 3 x 105 TC-1 cells. Statistically significant differences between the amphiphilic and soluble vaccine-treated groups are indicated by asterisks. ***, p<0.001; **, p<0.01; *, p<0.05. All data plotted as mean ± sem (n=3-8).

[0029] [Figure 6]Figure 6A is a bar graph showing OVA-specific CD8+ T cell % after treatment with free CpG and MPLA, lipo-G6-CpG-MPLA, or lipo-CpG-MPLA. Figure 6B is a bar graph showing TNF-α and INF-γ positive CD8+ T cell % after treatment with free CpG and MPLA, lipo-G6-CpG-MPLA, or lipo-CpG-MPLA. Figure 6C is a line graph showing OVA-specific CD8+ T cell % over time after treatment with free CpG and MPLA, lipo-G6-CpG-MPLA, or lipo-CpG-MPLA. Figure 6D is a bar graph showing OVA-specific CD8+ T cell % in blood, spleen, and lymph node after treatment with free CpG and MPLA, lipo-G6-CpG-MPLA, or lipo-CpG-MPLA.

[0030] [Figure 7-1] Figure 7A is a representative schematic of an exemplary micelle formed by self-assembly of an immunostimulatory conjugate showing a G-quadruplex structure formed by Hoogsteen hydrogen bonding. Figure 7B is a graph showing the size profile (diameter (nm)) of the self-assembled micelles. Figure 7C is a line graph showing the results of circular dichroism analysis (CD(mdeg)) of G-quadruplex-stabilized micelles in 1x PBS / 20 mM K+.

[0031] [Figure 7-2] Figure 7A is a representative schematic of an exemplary micelle formed by self-assembly of an immunostimulatory conjugate showing a G-quadruplex structure formed by Hoogsteen hydrogen bonding. Figure 7B is a graph showing the size profile (diameter (nm)) of the self-assembled micelles. Figure 7C is a line graph showing the results of circular dichroism analysis (CD(mdeg)) of G-quadruplex-stabilized micelles in 1x PBS / 20 mM K+.

[0032] [Figure 8-1]Figure 8A is a bar graph showing the quantitative accumulation (radiative efficiency) of various CpG-based micelles in the inguinal lymph nodes (left half of the graph) and axillary lymph nodes (right half of the graph) 24 hours after injection. Figure 8B is a bar graph showing the quantitative accumulation (radiative efficiency) of various CpG-based micelles in the inguinal lymph nodes (left half of the graph) and axillary lymph nodes (right half of the graph) 72 hours after injection. [Figure 8-2] Figure 8A is a bar graph showing the quantitative accumulation (radiative efficiency) of various CpG-based micelles in the inguinal lymph nodes (left half of the graph) and axillary lymph nodes (right half of the graph) 24 hours after injection. Figure 8B is a bar graph showing the quantitative accumulation (radiative efficiency) of various CpG-based micelles in the inguinal lymph nodes (left half of the graph) and axillary lymph nodes (right half of the graph) 72 hours after injection.

[0033] [Figure 9] FIG. 9 is a representative schematic diagram of a lipid-peptide conjugate.

[0034] [Figure 10] Figures 10A-10D are bar graphs showing the results of milliplex analyses of proinflammatory cytokines (IFN-γ in Figure 10A; TNF-α in Figure 10B; IL6 in Figure 10C; IL12p40 in Figure 10D) induced in peripheral blood of mice immunized with a single dose (6.2 nmol) of CpG formulation. Blood samples were collected at different time intervals (2 h and 24 h) and analyzed according to the manufacturer's instructions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Detailed Description of the Invention (I. Definition) An immunostimulatory oligonucleotide, as used herein, is an oligonucleotide that can stimulate (eg, induce or enhance) an immune response.

[0036] As used herein, CG oligodeoxynucleotides (CG ODNs) are short, single-stranded synthetic DNA molecules that contain a cytosine nucleotide (C) followed by a guanine nucleotide (G).

[0037] By "immune cell" is meant a cell of hematopoietic origin that plays a role in the immune response. Immune cells include lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes).

[0038] The term "T cells" refers to CD4+ T cells or CD8+ T cells. The term T cells includes TH1 cells, TH2 cells and TH17 cells.

[0039] The term "T cytotoxicity" includes any immune response mediated by CD8+ T cell activation. Exemplary immune responses include cytokine production, CD8+ T cell proliferation, granzyme or perforin production, and clearance of infectious agents.

[0040] Generally, as used herein, "pharmacologically acceptable" refers to those compounds, substances, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues, organs, and / or body fluids of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0041] The terms "subject," "individual," and "patient" refer to any individual who is a target of treatment using the disclosed compositions. The subject may be a vertebrate, e.g., a mammal. Thus, the subject may be a human. The subject may be symptomatic or asymptomatic. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. The subject may include a control subject or a test subject.

[0042] As used herein, the term "polypeptide" refers to a chain of amino acids of any length, regardless of modification (eg, phosphorylation or glycosylation).

[0043] The term "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to provide treatment for the disorder, disease, or condition being treated, to induce or enhance an immune response, or otherwise provide the desired pharmacological and / or physiological effect. The exact dosage will vary according to a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, the stage of the disease, and the treatment being performed.

[0044] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans, rodents (e.g., mice and rats), and other laboratory animals.

[0045] The term "oligonucleotide" or "polynucleotide" refers to a synthetic or isolated nucleic acid polymer containing multiple nucleotide subunits.

[0046] II. COMPOSITIONS Structural features of lipid conjugates were discovered that control the targeting of the conjugates to lymph nodes. Under physiological conditions, amphiphilic lipid conjugates exist in a three-way equilibrium as shown in Figure 2F. In pure water, certain lipid conjugates form micelles, whereas in the presence of serum and cells, these amphiphiles reach an equilibrium between binding to albumin and inserting their lipophilic tails into the cell membrane.

[0047] As discussed in more detail below, lipid conjugates that efficiently target lymph nodes typically contain three domains: a lipophilic domain that binds to albumin, a polar block domain, and a cargo such as a molecular adjuvant or immunostimulatory compound (e.g., an oligonucleotide) or an antigenic peptide. Depending on the cargo, the length and composition of the polar block can be tailored to push the equilibrium toward albumin binding, stable micelle formation, or cellular entry. The design guidelines and compositions disclosed below can be used to induce or enhance a strong immune response with low systemic toxicity because the immunostimulatory compound is localized to lymph nodes (i.e., lymph node-targeted conjugates) or tissues at the local site of administration (i.e., micelle-stabilized conjugates).

[0048] The effectiveness of any particular lipid conjugate for targeting lymph nodes can be assayed based on the ability of albumin to destroy the micelles formed by a plurality of the conjugates in aqueous solution.For example, if an albumin-containing agent (e.g., fetal bovine serum) can destroy 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more of the micelles formed in aqueous solution, the conjugate can be selected to target lymph nodes.However, if 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more of the micelles formed in aqueous solution remain intact in the presence of albumin, the conjugate can be selected as a micelle-stabilizing conjugate.

[0049] A. Lymph Node Targeting Conjugates Lipid conjugates, such as lipid-oligonucleotide and lipid-peptide conjugates for use in immunogenic compositions, are disclosed. Lymph node targeting conjugates can be transported from the administration site through lymph to lymph nodes where they accumulate and activate immune cells. It is believed that efficient lymph node accumulation of lipid conjugates depends on the ability of the amphiphile to partition from micelles to serum protein-bound state.

[0050] Lymph node targeting conjugates typically contain three domains: a very lipophilic albumin binding domain (e.g., an albumin binding lipid), a cargo (e.g., a molecular adjuvant or a peptide antigen), and a polar block linker that promotes the solubility of the conjugate and reduces the ability of the lipid to penetrate into the cell plasma membrane. Thus, in some embodiments, the general structure of the conjugate is LPC, where "L" is an albumin binding lipid, "P" is a polar block, and "C" is a cargo (e.g., a molecular adjuvant or a polypeptide). In some embodiments, the cargo itself can also function as a polar block domain, and a separate polar block domain is not required. Thus, in some embodiments, the conjugate is only two domains: an albumin binding lipid and a cargo. For example, lipid-oligonucleotide conjugates can contain an immunostimulatory oligonucleotide that is directly conjugated to a lipid or linked to a linker that is conjugated to a lipid. The lipid-peptide conjugate can comprise an antigenic peptide directly conjugated to a lipid or linked to a linker that is conjugated to a lipid.

[0051] Lipid-conjugated peptides are well known as vaccine agents (lipopeptides), whereas lipids directly conjugated to peptides do not exhibit lymph node targeting because the conjugates are not soluble enough to preferentially partition into albumin in the presence of cells; they instead tightly integrate into cell membranes and thus remain trapped at the injection site.

[0052] Antigenic peptides directly conjugated to lipids (lipopeptides) have been extensively studied as a modality to enhance vaccine efficacy (Jackson, et al in New Generation Vaccines (2011); Eriksson & Jackson Curr Protein Pept Sci 8, 412-417 (2007); BenMohamed, et al. The Lancet Infectious Diseases 2, 425-431 (2002)). These molecules generally do not exhibit lymph node targeting. This is exemplified by the data in Figures 2G and 2H, where it is shown that a very short PEG linker attached to an albumin-binding diacyl tail results in strong cell membrane penetration in vitro (2G) and accumulates to only a small extent in lymph nodes in vivo after subcutaneous injection (2H). Furthermore, peptide antigens directly linked to albumin-binding diacyl tails elicited little detectable immune response in vivo, whereas lipo-PEG-peptides elicited strong T cell responses (Figure 5C). A second difference from previously reported lipopeptides is that the diacyl tails that facilitate lipid binding and lymph node targeting do not exhibit direct adjuvant activity by themselves, unlike lipopeptides such as pam3cys-peptide conjugates that are known to have adjuvant activity via binding to TLR-2 and other immune stimulatory receptors.

[0053] (1. Lipids) The lipid conjugates disclosed herein typically include a hydrophobic lipid. The lipid may be linear, branched, or cyclic. The lipid is preferably at least 17-18 carbons in length, but may be shorter if it exhibits good albumin binding and adequate targeting to lymph nodes.

[0054] Lymph node targeting conjugates include lipid-oligonucleotide conjugates and lipid-peptide conjugates that can be transported from delivery site through lymph to lymph node. In a preferred embodiment, the activity depends in part on the ability of the conjugate to associate with albumin in the blood of the subject. Thus, lymph node targeting conjugates typically include lipids that can bind to albumin under physiological conditions. Suitable lipids for targeting lymph nodes can be selected based on the ability of the lipid or lipid conjugates that include the lipid to bind to albumin. Suitable methods for testing the ability of the lipid or lipid conjugate to bind to albumin are known in the art and are discussed in the following examples.

[0055] For example, in one embodiment, a plurality of lipid conjugates are allowed to spontaneously form micelles in an aqueous solution. The micelles are incubated with albumin or a solution containing albumin, such as fetal bovine serum (FBS). Samples can be analyzed, for example, by ELISA, size exclusion chromatography, or other methods, to determine whether binding has occurred, as illustrated in FIG. 2B. A lipid conjugate can be selected as a lymph node targeting conjugate if, in the presence of a solution containing albumin, such as albumin or fetal bovine serum (FBS), the micelles dissociate and the lipid conjugate binds to albumin as discussed above.

[0056] Examples of preferred lipids for use in lymph node-targeting lipid conjugates include, but are not limited to, fatty acids having an 8-30 carbon aliphatic tail (including, but not limited to, linear unsaturated and saturated fatty acids, branched saturated and unsaturated fatty acids, and fatty acid derivatives (e.g., fatty acid esters, fatty acid amides, and fatty acid thioesters)), diacyl lipids, cholesterol, cholesterol derivatives, and steroid acids (e.g., bile acids); lipid A, or combinations thereof.

[0057] In some embodiments, the lipid is a diacyl lipid or a two-tailed lipid. In some embodiments, the tail in the diacyl lipid contains about 8 to about 30 carbons and can be saturated, unsaturated, or a combination thereof. The tail can be attached to the head group via an ester bond linkage, an amide bond linkage, a thioester bond linkage, or a combination thereof. In certain embodiments, the diacyl lipid is a phospholipid, a glycolipid, a sphingolipid, or a combination thereof.

[0058] Preferably, the lymph node targeting conjugate comprises a lipid that is 8 or more carbon units in length. It is believed that increasing the number of lipid units may reduce the entry of the lipid into the plasma membrane of cells, allowing the lipid conjugate to remain free to bind to albumin and transport to the lymph nodes.

[0059] For example, the lipid can be a diacyl lipid composed of two C18 hydrocarbon tails.

[0060] In some embodiments, the lipid for use in preparing lymph node-targeting lipid conjugates is neither a single-chain hydrocarbon (e.g., C18) nor cholesterol. Cholesterol conjugates have been investigated to enhance the immunomodulation of molecular adjuvants (e.g., CpG) and the immunogenicity of peptides, but cholesterol conjugates associate well with lipoproteins but poorly with albumin, and show poor lymph node targeting and low immunogenicity in vaccines compared to optimal albumin-bound conjugates (Figure 2C).

[0061] (2. Cargo) The cargo of the conjugates disclosed herein is typically a molecular adjuvant, such as an immunostimulatory oligonucleotide, or a peptide antigen, but the cargo can also be other oligonucleotides, peptides, Toll-like receptor agonists or other immunomodulatory compounds, dyes, MRI contrast agents, fluorophores, or small molecule drugs that require efficient transport to lymph nodes.

[0062] (a. Molecular Adjuvants) Lipid-oligonucleotide conjugates are disclosed. The oligonucleotide conjugates described herein typically include immunostimulatory oligonucleotides.

[0063] In some embodiments, the immunostimulatory oligonucleotide can function as a ligand of pattern recognition receptor (PRR). An example of PRR is the Toll-like family of signaling molecules, which play a role in the initiation of innate immune response and also affect the later more antigen-specific adaptive immune response. Thus, the oligonucleotide can function as a ligand of Toll-like family signaling molecules, such as Toll-like receptor 9 (TLR9).

[0064] For example, unmethylated CpG sites can be detected by TLR9 on plasmacytoid dendritic cells and B cells in humans (Zaida, et al., Infection and Immunity, 76(5):2123-2129, (2008)). Thus, the sequence of the oligonucleotide can contain one or more unmethylated cytosine-guanine (CG or CpG (used interchangeably)) dinucleotide motifs. The "p" above refers to the phosphodiester backbone of DNA, as discussed in more detail below, and some oligonucleotides that contain CG can have modified backbones, such as phosphorothioate (PS) backbones.

[0065] In some embodiments, the immunostimulatory oligonucleotide may contain more than one CG dinucleotide, either consecutively or separated by intervening nucleotides.The CpG motif may be present inside the oligonucleotide sequence.Many nucleotide sequences stimulate TLR9 due to variations in the number and position of CG dinucleotides, and the exact base sequence adjacent to the CG dimer.

[0066] Typically, CG ODNs are classified based on their sequence, secondary structure, and effect on human peripheral blood mononuclear cells (PBMCs). The five classes are class A (type D), class B (type K), class C, class P, and class S (Vollmer, J & Krieg, AM, Advanced drug delivery reviews 61(3): 195-204 (2009) (hereby incorporated by reference). CG ODNs can stimulate the production of type I interferons (e.g., IFNα) and induce the maturation of dendritic cells (DCs). Some classes of ODNs are also potent activators of natural killer (NK) cells through indirect cytokine signaling. Several classes are potent stimulators of human B cell and monocyte maturation (Weiner, GL, PNAS USA 94(20): 10833-7 (1997); Dalpke, AH, Immunology 106(1): 102-12 (2002); Hartmann, G, J of Immun. 164(3):1617-2 (2000), each of which is incorporated herein by reference).

[0067] Other PRR Toll-like receptors include TLR3 and TLR7, which can recognize double-stranded RNA, single-stranded RNA, and short double-stranded RNA, respectively, as well as retinoic acid-inducible gene I (RIG-I)-like receptor (i.e., RIG-I) and melanoma differentiation-associated gene 5 (MDA5), which are best known as RNA-sensing receptors in the cytosol. Thus, in some embodiments, the oligonucleotide comprises a functional ligand of TLR3, TLR7, or RIG-I-like receptor, or a combination thereof.

[0068] Examples of immune stimulatory oligonucleotides and methods for making them are known in the art. See, e.g., Bodera, P. Recent Pat Inflamm Allergy Drug Discov. 5(1):87-93 (2011), incorporated herein by reference.

[0069] In some embodiments, the oligonucleotide cargo comprises two or more immunostimulatory sequences.

[0070] The oligonucleotides can be between 2 and 100 nucleotide bases in length (e.g., 5 nucleotide bases in length, 10 nucleotide bases in length, 15 nucleotide bases in length, 20 nucleotide bases in length, 25 nucleotide bases in length, 30 nucleotide bases in length, 35 nucleotide bases in length, 40 nucleotide bases in length, 45 nucleotide bases in length, 50 nucleotide bases in length, 60 nucleotide bases in length, 70 nucleotide bases in length, 80 nucleotide bases in length, 90 nucleotide bases in length, 95 nucleotide bases in length, 98 nucleotide bases in length, 100 nucleotide bases in length or more).

[0071] The 3' or 5' end of the oligonucleotide may be conjugated to the polar block or the lipid. In a preferred embodiment, the 5' end of the oligonucleotide is linked to the polar block or the lipid.

[0072] The oligonucleotides may typically be DNA or RNA nucleotides that contain a heterocyclic base (nucleobase), a sugar moiety attached to the heterocyclic base, and a phosphate moiety that esterifies the hydroxyl functional group of the sugar moiety. The majority of naturally occurring nucleotides contain uracil, thymine, cytosine, adenine, and guanine as heterocyclic bases, and ribose or deoxyribose sugars linked by phosphodiester bonds.

[0073] In some embodiments, the oligonucleotide is composed of nucleotide analogs that are chemically modified to improve stability, half-life, or specificity or affinity to target receptors compared to their DNA or RNA counterparts. Chemical modifications include chemical modifications of nucleobases, sugar moieties, nucleotide bonds, or combinations thereof. As used herein, "modified nucleotide" or "chemically modified nucleotide" defines a nucleotide that has chemical modifications of one or more of the components, such as the heterocyclic base, sugar moiety, or phosphate moiety. In some embodiments, the charge of the modified nucleotide is reduced compared to the DNA or RNA oligonucleotide of the same nucleobase sequence. For example, the oligonucleotide can have a low negative charge, no charge, or a positive charge.

[0074] Typically, nucleoside analogs support bases that can hydrogen bond by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner that allows such hydrogen bonding in a sequence-specific manner between the oligonucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or DNA). In some embodiments, the analogs have a substantially uncharged, phosphorus-containing backbone.

[0075] (i. Heterocyclic Bases) The main naturally occurring nucleotides include uracil, thymine, cytosine, adenine and guanine as heterocyclic bases. The oligonucleotides may contain chemical modifications to their nucleobase components. Chemical modifications of heterocyclic bases or heterocyclic base analogs may be effective to increase binding affinity or stability in binding target sequences. Chemically modified heterocyclic bases include, but are not limited to, inosine, 5-(1-propynyl)uracil (pU), 5-(1-propynyl)cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5 and 2-amino-5-(2'-deoxy-β-D-ribofuranosyl)pyridine (2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine derivatives. Cyclic dinucleotides known to trigger cytosolic danger sensors (eg, STING) can be used.

[0076] (ii. Sugar Modifications) Oligonucleotides may also contain nucleotides with modified sugar moieties or sugar moiety analogs. Sugar moiety modifications include, but are not limited to, 2'-O-aminoethoxy, 2'-O-aminoethyl (2'-OAE), 2'-O-methoxy, 2'-O-methyl, 2-guanidoethyl (2'-OGE), 2'-O,4'-C-methylene (LNA), 2'-O-(methoxyethyl) (2'-OME) and 2'-O-(N-(methyl)acetamido) (2'-OMA). Substitutions of 2'-O-aminoethyl sugar moieties are particularly preferred because they are protonated at neutral pH, suppressing charge repulsion between the TFO and the target duplex. This modification stabilizes the C3'-endo conformation of the ribose or deoxyribose and also forms a bridge with the i-1 phosphate in the purine strand of the duplex.

[0077] In some embodiments, the oligonucleotide is a morpholino oligonucleotide.Morpholino oligonucleotides are typically composed of two or more morpholino monomers that contain purine or pyrimidine base pairing moieties that are effective for binding to bases in polynucleotides by base-specific hydrogen bonding, and the morpholino monomers are linked together by phosphorus-containing bonds (1-3 atoms long) that connect the morpholino nitrogen of one monomer to the 5' exocyclic carbon of an adjacent monomer.The purine or pyrimidine base pairing moieties are typically adenine, cytosine, guanine, uracil or thymine. The synthesis, structure, and binding properties of morpholino oligomers are detailed in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337.

[0078] Important properties of the morpholino-based subunits typically include: the ability to be linked in oligomeric form by stable, uncharged backbone linkages; the ability to form polymers that have high T-terminal complementary bases with target nucleic acids (including target RNA); m the ability to support nucleotide bases (e.g., adenine, cytosine, guanine, thymidine, uracil, or inosine) such that they can hybridize to oligomers as short as 10-14 bases; the ability of such oligomers to be actively transported into mammalian cells; and the ability of the oligomer:RNA heteroduplex to resist RNAse degradation.

[0079] In some embodiments, oligonucleotides have base-pairing portions and use morpholino-based subunits joined by uncharged bonds as described above.

[0080] (iii. Internucleotide Bonds) Oligonucleotides are linked by internucleotide bonds, where internucleotide refers to the chemical bond between two nucleoside moieties. Modifications to the phosphate backbone of DNA or RNA oligonucleotides can increase the binding affinity or stability of oligonucleotides or reduce their susceptibility to nuclease digestion. Cationic modifications, including but not limited to diethyl-ethylenediamide (DEED) or dimethyl-aminopropylamine (DMAP), can be particularly useful due to the reduction of electrostatic repulsion between the oligonucleotide and the target. Modifications to the phosphate backbone can also include replacing one of the non-bridging oxygens in the phosphodiester bond with a sulfur atom. This substitution creates a phosphorothioate internucleoside bond instead of a phosphodiester bond. Oligonucleotides containing phosphorothioate internucleoside bonds have been shown to be more stable in vivo.

[0081] Examples of modified nucleotides with reduced charge include modified internucleotide linkages such as phosphate analogs with achiral and uncharged intersubunit linkages (e.g., Sterchak, EP et al., Organic Chem., 52:4202, (1987)), as well as uncharged morpholino-based polymers with achiral intersubunit linkages (see, e.g., U.S. Patent No. 5,034,506).Some internucleotide linkage analogs include morpholidates, acetals, and polyamide-linked heterocycles.

[0082] In another embodiment, the oligonucleotide is a locked nucleic acid. Locked nucleic acids (LNA) are modified RNA nucleotides (see, e.g., Braasch, et al., Chem. Biol., 8(1):1-7 (2001)). LNA forms hybrids with DNA that are more stable than DNA / DNA hybrids (properties similar to those of peptide nucleic acid (PNA) / DNA hybrids). Thus, LNA can be used just as PNA molecules are. LNA binding efficiency can be increased in some embodiments by adding a positive charge to it. Commercially available nucleic acid synthesizers and standard phosphoramidite chemistry can be used to make LNA.

[0083] In some embodiments, the oligonucleotide is composed of peptide nucleic acid.Peptide nucleic acid (PNA) is a synthetic DNA mimic in which the phosphate backbone of the oligonucleotide is replaced by repeating N-(2-aminoethyl)-glycine units throughout, and phosphodiester bonds are typically replaced by peptide bonds.Various heterocyclic bases are linked to the backbone by methylene carbonyl bonds.PNA maintains the spacing of heterocyclic bases similar to conventional DNA oligonucleotides, but is an achiral and neutrally charged molecule.Peptide nucleic acid is composed of peptide nucleic acid monomers.

[0084] Other backbone modifications include peptide and amino acid variations and modifications. Thus, the backbone components of oligonucleotides such as PNAs can be peptide bonds, or alternatively, they can be non-peptide bonds. Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as O-linkers), amino acids such as lysine (which are particularly useful when a positive charge is desired in the PNA), and the like. Methods for chemical assembly of PNAs are well known. See, for example, U.S. Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, and 5,786,571.

[0085] Oligonucleotides may optionally contain one or more terminal residues or modifications at either or both ends to increase the stability of said oligonucleotide and / or its affinity to its target.Commonly used positively charged moieties include the amino acids lysine and arginine, but other positively charged moieties may also be useful.Oligonucleotides may further be modified to be end-capped using propylamine groups to prevent degradation.Procedures for 3' or 5' capping oligonucleotides are well known in the art.

[0086] In some embodiments, the oligonucleotide is single-stranded DNA, single-stranded RNA, or double-stranded RNA.

[0087] (b. Peptide antigens) Lipid-peptide conjugates are disclosed. The peptide conjugates described herein typically include an antigenic protein or polypeptide.

[0088] The peptides can be 2-100 amino acids (aa), including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the peptides can be greater than 50 amino acids. In some embodiments, the peptides can be >100 amino acids.

[0089] The protein / peptide may be linear, branched or cyclic. The peptide may contain D amino acids, L amino acids, or a combination thereof. The peptide or protein may be conjugated to the polar block or lipid at the N-terminus or C-terminus of the peptide or protein.

[0090] The protein or polypeptide can be any protein or peptide that can induce or increase the ability of the immune system to generate antibody and T cell responses to the protein or peptide. Examples of specific peptide and protein antigens that can be used in the lipid-peptide conjugates disclosed herein are discussed in more detail below with respect to preferred antigens that can be used in vaccine formulations.

[0091] Lipid-protein based micelles can be formed in aqueous solution by self-assembly of a conjugate comprising a peptide antigen linked (bound) to a polyethylene glycol (PEG) moiety or a derivative or analogue thereof, which is linked to a hydrophobic lipid.

[0092] (c. Other Cargo) In general, the cargo may include a therapeutic, prophylactic, or diagnostic agent. For example, chemotherapeutic drugs are of interest for targeting tumors, since albumin is known to accumulate in tumors due to the EPR effect and also due to rapid metabolism in tumors.

[0093] In some embodiments, the lipid conjugates disclosed herein include a detection label, such as a fluorophore such as fluorescein or rhodamine, an Alexa Fluor dye, a DyLight Fluor dye, a Quasar and Cal Fluor dye, a cyanine dye (Cy3, Cy5, Cy5.5, Cy7) or other fluorescent dye. The label may be the cargo or may be attached to the cargo.

[0094] 3. Polar Blocks / Linkers The conjugate should remain soluble so that it can be efficiently transported to lymph nodes. Thus, a polar block linker can be included between the cargo and the lipid to increase the solubility of the conjugate. The polar block reduces or prevents the ability of the lipid to penetrate into the plasma membrane of cells (e.g., cells in tissues adjacent to the injection site). The polar block can also reduce or prevent the ability of the cargo (e.g., synthetic oligonucleotides containing a PS backbone) to nonspecifically associate with extracellular matrix proteins at the administration site. The polar block increases the solubility of the conjugate without interfering with its ability to bind to albumin. This combination of features is believed to allow the conjugate to bind to albumin present in serum or tissue fluids and remain in circulation until the albumin is transported to and retained in lymph nodes.

[0095] The length and composition of the polar block can be adjusted based on the selected lipid and cargo. For example, for oligonucleotide conjugates, the oligonucleotide itself can be sufficiently polar to ensure the solubility of the conjugate (e.g., oligonucleotides that are 10, 15, 20 or more nucleotides in length). Thus, in some embodiments, no additional polar block linker is required. However, depending on the amino acid sequence, some lipidated peptides can be essentially insoluble. In these cases, it may be desirable to include a polar block that mimics the effect of polar oligonucleotides.

[0096] Polar blocks can be used as part of any of the lipid conjugates described herein (e.g., lipid-oligonucleotide conjugates and lipid-peptide conjugates) to reduce cell membrane entry / preferential partitioning to albumin. Suitable polar blocks include, but are not limited to, oligonucleotides (e.g., those discussed above), hydrophilic polymers (including, but not limited to, poly(ethylene glycol) (MW: 500 Da to 20,000 Da), polyacrylamide (MW: 500 Da to 20,000 Da), polyacrylic acid); hydrophilic amino acid (e.g., serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof) chains; polysaccharides (including, but not limited to, dextran (MW: 1,000 Da to 2,000,000 Da)), or combinations thereof.

[0097] The hydrophobic lipid and the linker / cargo are covalently bonded. The covalent bond may be a non-cleavable bond or a cleavable bond. The non-cleavable bond may include an amide bond or a phosphate bond, and the cleavable bond may include a disulfide bond, an acid-cleavable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzyme-cleavable bond.

[0098] (i. Ethylene glycol linker) In preferred embodiments, the polar block is one or more ethylene glycol (EG) units, more preferably two or more EG units (i.e., polyethylene glycol (PEG)). For example, in some embodiments, a peptide conjugate comprises a protein or peptide (e.g., a peptide antigen) and a hydrophobic lipid linked by a polyethylene glycol (PEG) molecule or a derivative or analog thereof.

[0099] In some embodiments, the protein conjugates described herein comprise a protein antigen linked to PEG, which is subsequently linked to a hydrophobic lipid or lipid-Gn-ON conjugate, either covalently or via the formation of a protein-oligo conjugate that hybridizes to an oligomeric conjugate.

[0100] The exact number of EG units will depend on the lipid and the cargo, but typically the polar block can have between about 1 and about 100, between about 20 and about 80, between about 30 and about 70, or between about 40 and about 60 EG units. In some embodiments, the polar block has between about 45 and 55 EG units. For example, in one preferred embodiment, the polar block has 48 EG units.

[0101] (ii. Oligonucleotide Linker) As discussed above, in some embodiments, the polar block is an oligonucleotide. The liner of the polar block can have any sequence. For example, the sequence of the oligonucleotide can be a random sequence or a sequence specifically selected for its molecular or biochemical properties (e.g., highly polar). In some embodiments, the polar block linker comprises one or more consecutive series of adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogs thereof. In some embodiments, the polar block linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogs thereof.

[0102] In one embodiment, the linker is one or more guanines, for example, between 1 and 10 guanines. It has been discovered that varying the number of guanines between the cargo (e.g., CpG oligonucleotide) and the lipid tail controls micelle stability in the presence of serum proteins. Thus, the number of guanines in the linker can be selected based on the desired affinity of the conjugate for serum proteins (e.g., albumin). As illustrated in the examples below, when the cargo is a CpG immunostimulatory oligonucleotide and the lipid tail is a diacyl lipid, the number of guanines affects the ability of micelles formed in aqueous solution to dissociate in the presence of serum: Unstabilized micelles (Lipo-G0T 10 In the presence of guanine, 20% of the micelles (lipo-G2T8-CG) remained intact, while the remaining 80% were destroyed and bound to the FBS component. In the presence of guanine, the percentage of intact micelles increased from 36% (lipo-G2T8-CG) to 73% (lipo-G4T6-CG) and finally reached 90% (lipo-G6T4-CG). By increasing the number of guanines to 8 (lipo-G8T2-CG) and 10 (lipo-G 10 Increasing the concentration to T0-CG) did not further enhance micelle stability.

[0103] Thus, in preferred embodiments, the linker in the lymph node-targeting conjugate may contain 0, 1, or 2 guanines. As discussed in more detail below, linkers containing 3 or more consecutive guanines may be used to form micelle-stabilized conjugates with properties well suited for local application at or near the site of administration.

[0104] B. Micelle-Stabilized Conjugates Micelle-stabilized conjugates include conjugates such as lipid-oligonucleotide conjugates and lipid-peptide conjugates that accumulate in the tissue surrounding the delivery site. The conjugates typically do not bind to albumin. In some embodiments, the lipids used to prepare micelle-stabilized lipid conjugates are the same as those used in lymph node-targeting lipid conjugates discussed above, and the ability to withstand binding to albumin is controlled by the molecular or biochemical properties of the cargo, the linker, or a combination thereof. In some embodiments, lipids that are not effective for use in lymph node-targeting conjugates are useful in micelle-stabilized conjugates. Because the micelle-stabilized conjugates do not necessarily bind to albumin.

[0105] Micelle-stabilized conjugates can be selected based on their ability to spontaneously form micelles in aqueous solution that are not disrupted by serum components (e.g., albumin), as discussed above. Suitable methods for testing the ability of the lipid or lipid conjugate to bind to albumin are known in the art and are discussed in the Examples below. For example, in one embodiment, a plurality of lipid conjugates are capable of spontaneously forming micelles in aqueous solution. The micelles are incubated with albumin or a solution containing albumin (e.g., fetal bovine serum (FBS)). Samples can be analyzed, for example, by ELISA, size separation chromatography, or other methods to determine whether binding has occurred. A lipid conjugate can be selected as a micelle-stabilized conjugate if, in the presence of albumin or a solution containing albumin (e.g., fetal bovine serum (FBS)), the micelles remain intact and the lipid conjugate does not bind to albumin.

[0106] Examples of preferred lipids for use in the micelle-stabilized lipid conjugates include, but are not limited to, fatty acids having an 8-30 carbon aliphatic tail (including, but not limited to, linear unsaturated and saturated fatty acids, branched saturated and unsaturated fatty acids, and fatty acid derivatives (e.g., fatty acid esters, fatty acid amides, and fatty acid thioesters)), diacyl lipids, cholesterol, cholesterol derivatives, and steroid acids (e.g., bile acids); lipid A, or combinations thereof.

[0107] In some embodiments, the lipid is a diacyl lipid or a two-tailed lipid. In some embodiments, the tail in the diacyl lipid contains about 8 to about 30 carbons and can be saturated, unsaturated, or a combination thereof. The tail can be linked to the head group via an ester bond linkage, an amino bond linkage, a thioester bond linkage, or a combination thereof. In certain embodiments, the diacyl lipid is a phosphate lipid, a glycolipid, a sphingolipid, or a combination thereof.

[0108] As discussed above, in some embodiments, the stability of micelles in the presence of albumin is influenced by the linker. For example, the oligonucleotide (e.g., immunostimulatory oligonucleotide) and the lipid can be linked by three or more intervening guanine nucleotides. The nucleotides can be located at the 5' end of the oligonucleotide. The guanine-rich DNA sequence can form a quadruplex structure through hydrogen bonds, where an oligoguanine molecularly "glues" four individual guanine-rich DNA sequences together. Thus, the immunostimulatory oligonucleotide conjugate can self-assemble into a "G-quadruplex". This then assembles to form a micelle with a hydrophobic lipid core and a nucleic acid corona. As illustrated in the examples below, the dynamic stability of micelles can be controlled by varying the number of guanine nucleotides linking the hydrophobic lipid to the immunostimulatory oligonucleotide. In some embodiments, the immunostimulatory oligonucleotide and the hydrophobic lipid are linked by a single guanine at the 5' end of the oligonucleotide, while in other embodiments, the immunostimulatory oligonucleotide and the hydrophobic lipid are linked by a single guanine at the 5' end of the oligonucleotide. In some embodiments, the amino acid sequence is linked by two guanines at the ends. n ) contains 3 to 10 guanines (n = 3 to 10).

[0109] The cargo of the micelle-stabilized conjugate may include any of the cargoes discussed above with respect to lymph node-targeted conjugates, as well as small molecule, oligonucleotide, or peptide therapeutics (i.e., any cargo that one of skill in the art would select for accumulation at a local delivery site).

[0110] The micelle-stabilized conjugates can spontaneously form micelles in aqueous solutions by self-assembly. The micelles have a hydrophobic lipid core and a hydrophilic surface. The formation of micelles in an aqueous environment (e.g., water, buffer) is driven by hydrophobic interactions, and the micelles are stabilized by the formation of G-quadruplexes as described above. Micelles can be easily stabilized by the formation of cations (e.g., potassium (K + )) which further stabilizes the G-quadruplexes. The cations connect the two G-quadruplexes and minimize electrostatic interactions between the immunostimulatory oligonucleotides. Guanine-rich oligonucleotide sequences can fold into various types of structures (e.g., intramolecular, intermolecular, parallel, and antiparallel) (Davis, J. T. Angew. Chem. Int. Ed. Engl. 43, 668-698 (2004). To promote micellar self-assembly and minimize oligonucleotide folding, the lipid-oligonucleotide conjugates can be suspended in pure water to allow assembly of the micelles, and then a potassium-containing buffer can be added to stabilize the G-quadruplex.

[0111] In some embodiments, the micelles of a homogenous micelle population are substantially uniform in size. As used herein, a "homogeneous" population of micelles each contains the same type of lipid-oligonucleotide conjugate (e.g., L-5'-G n -CG-ODN-3' conjugate).

[0112] As discussed above, the stability of the micelle can be controlled by varying the number of guanine nucleotides in the polar block.For example, in some embodiments, the conjugate comprises one or more guanine nucleotides at the 5' end of the oligonucleotide and a hydrophobic lipid linked to the 5'-most guanine.Micelle "stability" as used herein refers to the resistance of the micelle to degradation or change in micelle size in the presence of serum, albumin, or other proteins or lipids, and / or to change in size or composition in the presence of cells.

[0113] The diameter of the micelles as described herein can be from about 3 nm to about 100 nm. In some embodiments, the diameter of the micelles is 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm , 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm, 90 nm, 91 nm, 92 nm, 93 nm, 94 nm, 95 nm, 96 nm, 97 nm, 98 nm, 99 nm, or 100 nm. In some embodiments, the diameter of the micelles is about 20 nm or about 50 nm.

[0114] III. FORMULATIONS A. Pharmaceutical Compositions Pharmaceutical compositions comprising lipid conjugates are provided.The pharmaceutical compositions can be for administration by parenteral route (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal route (either passively or by iontophoresis or electroporation), or transmucosal route (nasal, vaginal, rectal or sublingual) or by using biodegradable inserts, and can be formulated in dosage forms suitable for each administration route.

[0115] In some embodiments, the compositions are administered systemically, for example, by intravenous or intraperitoneal administration, in an amount effective to deliver the composition to targeted cells. Other possible routes include transdermal or oral.

[0116] In certain embodiments, the composition is administered locally, for example, by direct injection at the site to be treated. In some embodiments, the composition is injected or otherwise administered directly to one or more tumors. Typically, local injection results in an increase in the local concentration of the composition greater than can be achieved by systemic administration. In some embodiments, the composition is delivered locally to the appropriate cells by using a catheter or syringe. Other means of locally delivering such compositions to cells include using an infusion pump (e.g., from Alza Corporation, Palo Alto, Calif.) or incorporating the composition into a polymeric implant (see, e.g., P. Johnson and JG Lloyd-Jones, eds., Drug Delivery Systems (Chichester, England: Ellis Horwood Ltd., 1987)), which can result in the sustained release of nanolipogel in the area immediately adjacent to the implant.

[0117] As further research is conducted, information regarding appropriate dosage levels for the treatment of various conditions in various patients will become apparent, and those skilled in the art will be able to ascertain appropriate dosing, taking into account the context of the treatment, the recipient's age, and the general health of the patient. The selected dosage will depend on the desired therapeutic effect, the route of administration, and the desired duration of treatment. In general, dosage levels of 0.001-10 mg / kg body weight per day are administered to mammals. In general, for intravenous injection or infusion, the dosage may be less.

[0118] 1. Formulations for Parenteral Administration In preferred embodiments, the lipid conjugates are administered parenterally in an aqueous solution, hi some embodiments, the composition includes albumin or other serum proteins.

[0119] The formulations may be in the form of suspensions or emulsions. Generally, a pharmaceutical composition is provided containing an effective amount of the conjugate, and optionally includes pharma- ceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions may contain a variety of additives, such as diluents, sterile water, buffered saline of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and, optionally, additives such as surfactants and solubilizers (e.g., TWEEN® 20, TWEEN® 80, also known as polysorbate 20 or polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimerosal, benzyl alcohol) and bulking substances. The formulation may contain a glycerin, glycerol, sorbitol, sorbitol, sorbitol derivative, sorbitol derivatives, etc. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil and corn oil), gelatin, and injectable organic esters (e.g., ethyl oleate). The formulations may be lyophilized and redissolved / resuspended immediately prior to use. The formulations may be sterilized, for example, by filtering through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0120] 2. Formulations for Topical and Mucosal Administration The lipid conjugates may be applied topically. Topical administration may include application to the pulmonary, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa. In some cases, the conjugates may be transcytosed into albumin across the mucosal barrier.

[0121] The compositions, when delivered as either an aerosol or spray-dried particles having an aerodynamic diameter of less than about 5 microns, can be delivered to the lungs upon inhalation and cross through the lining of the lung epithelium into the bloodstream.

[0122] A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent® nebulizer (Mallinckrodt Inc., St. Louis, Mo.); Acorn® II nebulizer (Marquest Medical Products, Englewood, Colo.); Ventolin® metered dose inhaler (Glaxo Inc., Research Triangle Park, NC); and Spinhaler® powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes, and Mannkind all have inhalable insulin powder preparations approved or in clinical trials, where the above technology can be applied to the formulations described herein.

[0123] Formulations for administration to mucosa are typically spray-dried drug particles, which can be incorporated into tablets, gels, capsules, suspensions or emulsions. Standard pharmaceutical excipients are available from any formulator. Oral formulations can be in the form of chewing gum, gel strips, tablets, capsules, or lozenges.

[0124] Transdermal formulations can also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard techniques. Transdermal formulations can include penetration enhancers.

[0125] (B. Immunogenic composition) The conjugates disclosed herein can be used as components in immunogenic compositions or vaccines.Typically, the immunogenic compositions disclosed herein include an adjuvant, an antigen, or a combination thereof.The combination of an adjuvant and an antigen can be called a vaccine.When administered to a subject in combination, the adjuvant and the antigen can be administered in separate pharmaceutical compositions, or they can be administered together in the same pharmaceutical composition.When administered in combination, the adjuvant can be a lipid conjugate, the antigen can be a lipid conjugate, or both the adjuvant and the antigen can be lipid conjugates.

[0126] (1. Antigen) The immunogenic composition may include an adjuvant lipid conjugate (e.g., an immunostimulatory oligonucleotide-lipid conjugate) administered alone or in combination with an antigen. The antigen may be a peptide, protein, polysaccharide, saccharide, lipid, nucleic acid, or a combination thereof. The antigen may be derived from a virus, bacteria, parasite, plant, protozoan, fungus, tissue, or transformed cell (e.g., cancer or leukemia cell), and may be a whole cell or its immunogenic component (e.g., a cell wall component or its molecular component).

[0127] Suitable antigens are known in the art and commercially available from government and scientific institutions. In one embodiment, the antigen is an inactivated or attenuated whole organism. The organisms can be infectious organisms, such as viruses, parasites, and bacteria. The organisms can also be tumor cells. The antigen can be a purified or partially purified polypeptide from a tumor or a viral or bacterial source. The antigen can be a recombinant polypeptide produced by expressing DNA encoding a polypeptide antigen in a heterologous expression system. The antigen can be DNA encoding all or part of an antigenic protein. The DNA can be in the form of vector DNA, such as plasmid DNA.

[0128] Antigens can be provided as single antigens or in combination. Antigens can also be provided as complex mixtures of polypeptides or nucleic acids. Exemplary antigens are provided below.

[0129] (a. viral antigen) Viral antigens may be isolated from any virus, including, but not limited to, any of the following virus families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., coronaviruses such as Severe Acute Respiratory Syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire strain, Reston strain, Ivory strain, Coast or Sudan strains), Flaviviridae (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus 4), Hepadnaviridae, Herpesviridae(e.g., human herpesviruses 1, 3, 4, 5, and 6, and cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., influenza viruses A, B, and C), Papovaviridae, Paramyxoviridae (e.g., measles virus, mumps virus, and human respiratory syncytial virus), Parvoviridae, Picornaviridae, ae (e.g., poliovirus, rhinovirus, hepatovirus, and foot and mouth disease virus), Poxviridae (e.g., vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentiviruses such as human immunodeficiency virus (HIV) 1 and HIV2), Rhabdoviridae (e.g., rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (e.g., rubella virus, dengue virus, etc.), and Totiviridae. Suitable viral antigens also include all or part of dengue protein M, dengue protein E, dengue D1NS1, dengue D1NS2, and dengue D1NS3.

[0130] Viral antigens may be derived from specific strains (e.g., Papillomavirus, Herpesvirus, e.g., Herpes simplex 1 and 2; Hepatitis virus, e.g., Hepatitis A virus (HAV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis Delta (D) virus (HDV), Hepatitis E virus (HEV), and Hepatitis G virus (HGV), Tick-borne encephalitis virus; Parainfluenza virus, Varicella-Zoster virus, Cytomegalovirus, Epstein-Barr virus, Rotavirus, Rhinovirus, Adenovirus, Coxsackievirus, Equine encephalitis virus, Japanese encephalitis virus, Yellow fever virus, Rift Valley fever virus, and Lymphocytic choriomeningitis virus).

[0131] (b. Bacterial antigen) Bacterial antigens may be derived from any bacteria, including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Ricket tsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, and Yersinia.

[0132] (c.parasitic antigen) Parasitic antigens may be derived from parasites, including, but not limited to, antigens from Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni. These include all or part of sporozoan antigens, plasmodium antigens, such as circumsporozoite protein, sporozoite surface protein, liver stage antigen, apical membrane associated protein, or merozoite surface protein.

[0133] (d. Allergens and Environmental Antigens) The antigen may be an allergen or an environmental antigen, such as a naturally occurring allergen, including, but not limited to, pollen allergens (tree, herb, weed, and grass pollen allergens), insect allergens (inhalant, saliva, and venom allergens), animal hair and dander allergens, and food allergens. Important tree, grass, and herb pollen allergens are from the taxonomic orders Fagales, Oleales, Pinales, and platanaceae, particularly birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus), and olive (Olea), cedar (Cryptomeria and Juniperus, Platanus), Poales (including grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum), Asterales, and Urticales (including herbs of the genera Ambrosia, Artemisia, and Parietaria, among others). Other allergen antigens that may be used include dust mites of the genera Dermatophagoides and Euroglyphus, such as dust mites of the genera Lepidoglyphys, Glycyphagus, and Tyrophagus. allergens from insects, cockroaches, midges and fleas (e.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides), mammals (e.g. cats, dogs and horses), birds, venom allergens (including allergens from stinging and biting insects (e.g. from the taxonomic order Hymenoptera which includes bees (superfamily Apidae), wasps (superfamily Vespidea) and ants (superfamily Formicoidae)). Still other allergen antigens which may be used include inhalant allergens from fungi (e.g. from the genera Alternaria and Cladosporium).

[0134] (e.Cancer antigen) Cancer antigens are typically antigens that are preferentially expressed by cancer cells, i.e., expressed at higher levels in cancer cells than in non-cancerous cells, and in some cases, expressed only by cancer cells. The cancer antigens can be expressed within cancer cells or on the surface of cancer cells. The cancer antigens can be MART-1 / Melan-A, gp100, adenosine deaminase binding protein (ADAbp), FAP, cyclophilin b, colorectal-related antigen (CRC)--C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, and CD20. The cancer antigen may be selected from the group consisting of MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MU C family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus proteins, Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20, or c-erbB-2.

[0135] (2. Adjuvants) The immunogenic compositions can include antigenic lipid conjugates, such as antigenic polypeptide-lipid conjugates, administered alone or in combination with an adjuvant.

[0136] Such adjuvants include alum (e.g., aluminum hydroxide, aluminum phosphate); saponin purified from the bark of the Q. saponaria tree (e.g., QS21 (a glycolipid eluting in the 21st peak in HPLC fractionation; Antigenics, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS (immunostimulating complexes containing a mixture of saponin, lipid, and virus-sized particles with pores that can hold antigens; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beecham adjuvant system #4 containing alum and MPL; SBB, Belgium), CRL These can be, but are not limited to, micelle-forming non-ionic block copolymers such as 1005 (which contain linear chains of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene, Vaxcel, Inc., Norcross, Ga.), as well as Montanide IMS (e.g., IMS 1312, a water-based nanoparticle combined with the soluble immune stimulant Seppic).

[0137] The adjuvant may be a TLR ligand, such as those discussed above. Adjuvants that function through TLR3 include, but are not limited to, double-stranded RNA. Adjuvants that function through TLR4 include derivatives of lipopolysaccharides (e.g., monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont.)) and muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (glucosamine disaccharide related to lipid A; OM Adjuvants that function through TLR5 include, but are not limited to, flagellin. Adjuvants that function through TLR7 and / or TLR8 include single-stranded RNA, oligoribonucleotides (ORN), synthetic low molecular weight compounds (e.g., imidazoquinoline amines (e.g., imiquimod (R-837), resiquimod (R-848)). Adjuvants that function through TLR9 include DNA of viral or bacterial origin, or synthetic oligodeoxynucleotides (ODN) (e.g., CpG ODN). Another class of adjuvants are phosphorothioate-containing molecules (e.g., phosphorothioate nucleotide analogs) and nucleic acids that contain phosphorothioate backbone linkages.

[0138] The adjuvants can also be oil emulsions (e.g., Freund's adjuvant); saponin formulations; virosomes and virus-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; mineral-containing compositions (e.g., inorganic salts (e.g., aluminum and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and / or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester formulations; polyphosphazenes; muramyl peptides; imidazoquinolone compounds; and surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0139] Adjuvants can also include immune modulators, such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-γ), macrophage colony-stimulating factor, and tumor necrosis factor.

[0140] C. Combination Therapy In some embodiments, the conjugate is administered in combination with one or more additional therapeutic agents, which may be administered in the same pharmaceutical composition as the conjugate, or the conjugate and the additional therapeutic agent may be administered in separate pharmaceutical compositions.

[0141] In some embodiments, the conjugate is administered in combination with conventional therapeutic agents used for the treatment of the disease or condition to be treated.Conventional therapeutic agents are known in the art and can be determined by those skilled in the art based on the disease or disorder to be treated.For example, if the disease or condition is cancer, the conjugate can be co-administered with chemotherapy drugs; or if the disease or condition is bacterial infection, the conjugate can be co-administered with antibiotics.

[0142] (IV. Usage) A. Methods for Delivering Immunostimulants (1. Lymph node targeting) The data presented below support the discovery that conjugation of a cargo (e.g., an oligonucleotide or a peptide) to an albumin-binding domain can increase delivery and accumulation of the cargo to lymph nodes. Lymph nodes are ovoid organs of the immune system that are widely distributed throughout the body, including the axilla and stomach, and are connected by lymphatic vessels. Lymph nodes are home to B cells, T cells, and other immune cells. Lymph nodes act as filters or traps for foreign particles and are important in the proper functioning of the immune system. They are densely packed with lymphocytes and white blood cells called macrophages.

[0143] Lymph node-targeted conjugates are typically transported from the injection site to secondary lymphatic organs (e.g., lymph nodes) where they interact with immune cells. Albumin binding of the conjugates is believed to prevent them from rapidly shedding into the bloodstream, preventing them from being filtered, accumulating, and retargeting to lymphatic vessels and draining lymph nodes where they present their immunostimulatory oligonucleotides, antigenic peptides, or other cargo to immune cells.

[0144] As discussed above, albumin-bound lipids can be conjugated to the oligonucleotide or the antigenic peptide, which increases the immunostimulatory effect of the immunostimulatory oligonucleotide or the antigenic peptide, for example, compared to administering unconjugated oligonucleotide or antigenic peptide.In some embodiments, the conjugation of the immunostimulatory oligonucleotide or peptide antigen to albumin-bound lipid increases the accumulation of the cargo by 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more compared to unconjugated cargo.

[0145] (2.Tissue-specific targeting) Micelle-stabilized conjugates can be used to increase the delivery and accumulation of the cargo in tissues at or near the site of administration. Micelle-stabilized conjugates are believed to be resistant to destruction by serum proteins such as albumin. Thus, they can accumulate at the injection site, for example, by binding to extracellular matrix proteins or by penetrating into the cell membranes of local cells.

[0146] Micelle-stabilized conjugates can be used to increase the local accumulation of immunostimulatory oligonucleotides, antigenic peptides, small molecules, and other targets at the site of administration. In some embodiments, conjugates of the immunostimulatory oligonucleotide or peptide antigen increase the local accumulation of the cargo by 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, compared to the unconjugated cargo.

[0147] B. Methods for Increasing Immune Responses Lipid conjugates containing immunostimulatory oligonucleotides or antigenic peptide cargos can be administered in an amount effective to induce, increase or enhance an immune response. The "immune response" refers to a response that induces, increases or perpetuates the activation or effectiveness of innate or adaptive immunity. In addition, albumin-linked lipid conjugates of polypeptide antigens administered in the absence of other adjuvants can be used to promote tolerance rather than immunity to allergens or autoimmune antigens, for example. The conjugates can be delivered parenterally (by subcutaneous, intradermal or intramuscular injection) via lymphatics, or by systemic administration via the circulatory system. It has been shown that lymph nodes can filter conjugates bound to albumin. Thus, in some embodiments, parenteral administration does not result in systemic distribution, because the conjugates can be preferentially filtered by the nearest lymph nodes. This tendency also reduces systemic toxicity, such as splenic swelling.

[0148] Thus, in some embodiments, the conjugates may be administered to a site adjacent to or leading to one or more lymph nodes near the site of need for an immune response (i.e., near the tumor or site of infection). In some embodiments, the conjugates are administered in multiple doses to various locations throughout the body. The conjugates, particularly micelle-stabilized conjugates, may also be administered directly to the site of need for an immune response (e.g., tumor or site of infection).

[0149] The immune response may be induced, augmented, or enhanced by the Lipid conjugate compared to a control (e.g., a subject's immune response induced, augmented, or enhanced by cargo alone, or by the cargo being delivered using an alternative delivery strategy, such as liposomes). As discussed in more detail below, in some embodiments, the Lipid conjugate reduces inactivation and / or prolongs activation of T cells (i.e., increases antigen-specific proliferation of T cells, enhances cytokine production by T cells, stimulates differentiation and effector function of T cells, and / or promotes T cell survival), or overcomes T cell exhaustion and / or anergy.

[0150] The lipid conjugates can be used to induce immune responses, for example, when the cargo is administered alone or when the cargo is combined with an alternative delivery system and is not effective.The lipid conjugates can also be used to enhance or improve immune responses compared to administering cargo alone.In some embodiments, the lipid conjugates can reduce the dosage required to induce, increase or enhance immune responses; or can shorten the time required for the immune system to respond after administration.

[0151] The lipid conjugates can be administered as part of a prophylactic vaccine or immunogenic composition that confers resistance in a subject to subsequent exposure to an infectious agent, or as part of a therapeutic vaccine that can be used to initiate or enhance a subject's immune response to an existing antigen (e.g., a viral antigen in a subject infected with a virus or with cancer).

[0152] The desired outcome of a preventive or therapeutic immune response may vary according to the disease or condition to be treated or according to principles well known in the art. For example, an immune response against an infectious agent may completely prevent the colonization and replication of the infectious agent, resulting in "sterile immunity" and the absence of any disease symptoms. However, a vaccine against an infectious agent may be considered effective if it reduces the number, severity or duration of symptoms; reduces the number of individuals in a population with symptoms; or reduces the transmission of the infectious agent. Similarly, an immune response against cancer, an allergen, or an infectious agent may completely treat the disease, reduce symptoms, or be an aspect of an overall therapeutic intervention against the disease.

[0153] The lipid conjugate induces an improved effector cell response (e.g., a CD4 T cell immune response) against at least one of the constituent antigens or antigenic compositions, compared to the effector cell response obtained by the corresponding composition without the lipid conjugate. The term "improved effector cell response" refers to a higher effector cell response (e.g., a CD8 or CD4 response) obtained in a human patient after administration of the vaccine composition than that obtained after administration of the same composition without the lipid conjugate.

[0154] Improved effector cell responses can be obtained in immunologically unprimed patients, i.e., patients who are seronegative to the antigen. This seronegativity can be the result of patients who have never encountered the antigen (so-called "naive" patients) or, alternatively, patients who did not respond to an antigen once encountered. In some embodiments, the improved effector cell responses are obtained in immunocompromised subjects.

[0155] The improved effector cell response may be assessed by measuring the number of cells that produce any of the following cytokines: (1) cells that produce at least two different cytokines (CD40L, IL-2, IFN-γ, TNF-α); (2) cells that produce at least CD40L and another cytokine (IL-2, TNF-α, IFN-γ); (3) cells that produce at least IL-2 and another cytokine (CD40L, TNF-α, IFN-γ); (4) cells that produce at least IFN-γ and another cytokine (IL-2, TNF-α, CD40L); and (5) cells that produce at least TNF-α and another cytokine (IL-2, CD40L, IFN-γ).

[0156] An improved effector cell response is present when cells producing any of the above cytokines are more abundant following administration of the vaccine composition compared to a control as discussed above.

[0157] In a preferred embodiment, the composition increases the number of T cells that produce IFN-γ, TNF-α, or a combination thereof, or increases the production of IFN-γ, TNF-α, or a combination thereof in existing T cells.

[0158] In some embodiments, administration of the immunogenic composition alternatively or additionally induces an improved B memory cell response in patients receiving the lipid conjugate compared to a control. Improved B memory cell response is intended to mean an increase in the frequency of peripheral blood B lymphocytes that can differentiate into antibody-secreting plasma cells upon antigen encounter, as measured by stimulation of differentiation in vitro.

[0159] In yet another embodiment, the immunogenic composition enhances primary immune response as well as CD8 response. Administration of the lipid conjugate induces improved CD4 T cell or CD8 T cell immune response against a specific antigen compared to control. This method can induce a more sustained CD4 T cell response.

[0160] Preferably, the CD4 T cell immune response (e.g., an improved CD4 T cell immune response obtained in an unprimed subject) includes the induction of a cross-reactive CD4 T helper response. In particular, the amount of cross-reactive CD4 T cells is increased. The term "cross-reactive" CD4 response refers, for example, to CD4 T cells targeting epitopes shared between influenza strains.

[0161] C. The Disease to be Treated (1. Cancer) The disclosed lipid conjugates are useful for stimulating or enhancing the host immune response to treat cancer.The types of cancer that can be treated with the compositions and methods provided above include, but are not limited to, bladder, brain, breast, cervical, colorectal, esophageal, renal, liver, lung, nasopharyngeal, pancreatic, prostate, skin, stomach, uterus, ovary, testis and blood.

[0162] Malignant tumors that can be treated are classified herein according to the embryonic origin of the tissue from which the tumor originates. Carcinomas are tumors that arise from endodermal or ectodermal tissues (e.g., the skin or the epithelium lining of internal organs and glands). Sarcomas occur less frequently and originate from mesodermal connective tissues (e.g., bone, fat, and cartilage). Leukemias and lymphomas are malignant tumors of hematopoietic cells in the bone marrow. Leukemias grow as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors can appear and establish cancer in many organs or tissues of the body.

[0163] The conjugates can be administered as an immunogenic composition or as part of a vaccine, such as a prophylactic vaccine or a therapeutic vaccine that can be used to initiate or enhance a subject's immune response to an existing antigen, such as a tumor antigen in a subject with cancer.

[0164] The desired outcome of the preventive or therapeutic immune response may vary according to the disease, according to principles well known in the art.Similarly, the immune response against cancer may alleviate symptoms or be an aspect of the overall therapeutic intervention against the disease.For example, administration of the lipid conjugate may reduce tumor size or slow tumor growth compared to control.The stimulation of the immune response against cancer may be combined with surgical, chemotherapy, radiology, humoral immunology and other immunological approaches to affect treatment.

[0165] (2. Infectious diseases) In a preferred embodiment, the lipid conjugate is useful for treating acute or chronic infectious diseases. Since viral infections are primarily cleared by T cells, increasing T cell activity is therapeutically useful in situations where more rapid or complete clearance of infectious viral agents is beneficial to animals or human subjects. Thus, the lipid conjugate antagonist can be administered for the treatment of local or systemic viral infections, including but not limited to viral infections such as immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), and cold (e.g., human rhinovirus). For example, pharmaceutical formulations containing the lipid conjugate can be administered locally to treat viral skin diseases (e.g., herpes lesions or shingles), or genital warts. The lipid conjugate can also be administered to treat systemic viral diseases, including but not limited to AIDS, influenza, cold, or encephalitis.

[0166] It is also a smooth processor and it is fully suitable for Actino myces、Anabaena、Bacillus、Bacteroides、 Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Ch lorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Haemophilus influenza タイプB(HIB)、Histoplasma、Hyphomicrobium、Legionella、Leishmania、Leptspirosis、Listeria、Meningococcus A、BおよびC、Methanobacterium、Micrococcus、Myobacterium、Mycoplasma、Myxococcus、Neisseria 、Nitrobacter、Oscillatoria、Prochloron、Proteus、Pseudomonas、Phodospirillum、Rickettsia 、Salmonella、Shigella、Spirillum、Spirochaeta、Staphylococcus、Streptococcus、Streptomyc es, Sulfolobus, Thermoplasma, Thiobacillus, Treponema, Vibrio, Yersinia, and Cryptococcus neoformans、Histoplasma capsulatum、Candida albicans、Candida tropicalis、Nocardia asteroides、Rickettsia ricketsii、Rickettsia In particular, the present invention is directed to infections caused by microorganisms including, but not limited to, Mycoplasma typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Plasmodium vivax, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni.

[0167] In certain embodiments, the type of disease being treated or prevented is a chronic infectious disease caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens that invade cells and are attacked, for example, by cytotoxic T lymphocytes.

[0168] In preferred embodiments, the infection being treated is a chronic infection caused by a hepatitis virus, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes virus, Epstein-Barr virus, or human papilloma virus. EXAMPLES

[0169] Example 1: Albumin-bound lipo-oligoconjugates accumulate in lymph nodes (material and method) (Oligonucleotide synthesis) Oligonucleotides were synthesized on an automated DNA synthesizer (ABI 394, Applied Biosystems, Inc.) at a 1.0 micromolar scale. All DNA synthesis reagents, including cholesteryl-TEG phosphoramidite and DMT-PEG-phosphoramidite, were purchased from Glenres and Chemgenes and used according to the manufacturer's instructions. The immunostimulatory CpG oligo used was the type B sequence known as 1826. Synthesis and solid-phase conjugation of lipid phosphoramidites followed previous reports. Particle size was determined by dynamic light scattering (DLS) using a 90Plus / ZetaPals particle size and ξ potential analyzer (Brookhaven Instruments). DSPE-PEG 2000 -maleimide was purchased from Laysan Bio Inc. Carboxyfluorescein-labeled PEG 2000 -DSPE was purchased from Avanti Polar lipids Inc. Carboxyfluorescein-labeled NHS-PEG 2000 was purchased from nanocs Inc. Peptides were purchased from Genscript Corp. (Piscataway, NJ). Incomplete Freund's adjuvant (IFA) and fatty acid-free BSA were purchased from Sigma-Aldrich.

[0170] (Synthesis of diacyl lipid phosphoramidites) [ka]

[0171] Diacyl lipid phosphoramidites were prepared as described by Liu, et al. J. Angew. It was synthesized in two steps as described by E. et al., Chem., Int. Ed. 2011, 50, 7252-7255.

[0172] A solution of stearoyl chloride (6.789 g, 22.41 mmol) in ClCH2CH2Cl (50 ml) was added dropwise to a solution of 1,3-diamino-2-dydroxypropane (1.0 g, 11.10 mmol) in the presence of ClCH2CH2Cl (100 ml) and triethylamine (2.896 g, 22.41 mmol). The reaction mixture was stirred at room temperature for 2 hours and then heated at 70° C. overnight. The reaction mixture was then cooled to room temperature, filtered, and the solid was washed with CH2Cl2, CH3OH, 5% NaHCO3, and diethyl ether, respectively. The solid was dried under vacuum to give the intermediate product as a white solid (yield: 90%). 1 H NMR (300 MHz, CDCl3): δ 6.3 (m, 2H), 3.8 (m, 1H), 3.4-3.2 (m, 4H), 2.2 (t, 4H), 1.6 (m, 4H), 1.3-1.2 (m, 60H), 0.9 (t, 6H). The above intermediate product (5.8 g, 9.31 mmol) and DIPEA (4.2 mL, 18.62 mmol) were then dissolved in anhydrous CHCl2 (100 ml). The solution was cooled in an ice bath and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (8.6 mL, 0.47 mmol) was added dropwise under dry nitrogen. After stirring at room temperature for 1 h, the solution was heated to 60 °C for 90 min. The reaction mixture was washed with 5% NaHCO3 and brine, dried over Na2SO4, and concentrated under vacuum. The final product was isolated by precipitation from acetone to give 4 g (55% yield) of the phosphoramidite as a white solid. 1 H NMR (300 MHz, CDCl3): δ 6.4 (m, 2H), 3.9 (m, 2H), 3.8 (m, 2H), 3.6 (m, 2H), 3.0-2.9 (m, 2H), 2.6 (t, 2H), 2.2 (m, 4H), 1.6 (m, 6H), 1.3-1.2 (m, 72H), 0.9 (t, 6H). 31 P NMR (CDCl3) 154 ppm.

[0173] (DNA synthesis and lipophilic conjugation) All DNA and RNA sequences were synthesized on a 1.0 μM scale using an ABI 394 synthesizer. All lipophilic phosphoramidites were conjugated as the last "base" at the 5' end of the oligos. Lipophilic phosphoramidites were dissolved in dichloromethane and synthesized using the so-called syringe synthesis technique (Storhoff, et al., J Am. Chem. Soc., 120:1959-1964 (1999)). Briefly, lipid phosphoramidites (200 μL) were mixed with an activator (0.2 mM 5-ethylthiotetrazole in 200 μL acetonitrile) and the mixture was pushed back and forth between the CpG columns for 10 min using two syringes. Alternatively, lipophilic phosphoramidites can also be coupled using the DNA synthesizer (15 min coupling time). After synthesis, DNA was cleaved from the CpG, deprotected and purified by reversed-phase HPLC using a C4 column (BioBasic-4, 200 mm×4.6 mm, Thermo Scientific) and 100 mM triethylamine-acetate buffer (TEAA, pH 7.5)-acetonitrile (0-30 min, 10-100%) as eluent. Lipophilic ODN typically eluted at 20 min, while unconjugated ODN eluted at 8 min. The immunostimulatory CpG oligo used was a type B sequence known as 1826 (Ballas, et al., J. Immunol., 167, 4878-4886 (2001).

[0174] Lipo-G n Representative sequences of CpG: [ka]

[0175] (Synthesis of pyrene phosphoramidite) [ka]

[0176] Synthesis of Compound 1: In a 300ml round bottom flask, D-threoninol (0.95g, 9.1mmol), 1-pyrenebutyric acid (2.88g, 10.0mmol), DCC (2.06g, 10.0mmol) and NHS (1.15g, 10mmol) were dissolved in 50ml DMF. The reaction mixture was stirred at room temperature for 24 hours. Insoluble N,N'-dicyclohexylurea was filtered and DMF was removed by rotary vacuum evaporator to obtain oily crude product. Compound 1 was purified by flash chromatography (yield: 85%). 1H NMR (300 MHz, CDCl3): δ 8.1-7.7 (m, 9H), 6.2 (d, 1H), 4.2-3.8 (m, 4H), 3.0 (m, 2H), 2.3-2.2 (m, 4H), 1.2 (d, 3H).

[0177] Synthesis of compound 2: Compound 1 (2.93 g, 7.2 mmol) and 4-dimethylaminopyridine (0.043 g, 0.36 mmol) in 40 ml dry pyridine were placed in a 100 ml round bottom flask under dry nitrogen. The solution was cooled in an ice bath. DMT-Cl (2.93 g, 8.64 mmol) was dissolved in 10 ml dry CHCl in a 50 ml flask under nitrogen and slowly added to the pyridine solution under dry nitrogen. The reaction was allowed to slowly warm to room temperature and stirred for 24 hours. The solvent was removed under vacuum and compound 2 was isolated by chromatography (50:50:3 ethyl acetate:hexane / triethylamine) (yield: 75%). 1H NMR (300 MHz, CDCl3): δ 8.3-7.5 (m, 22H), 6.1 (d, 1H), 4.2-3.9 (m, 2H), 3.7 (d, 6H), 3.4-3.3 (m, 4H), 2.4-2.2 (m, 4H), 1.2 (d, 3H).

[0178] Synthesis of compound 3: Compound 2 (1 g, 1.48 mmol) was dissolved in CH2Cl2 and cooled in an ice bath. Then, DIPEA (0.57 g, 4.44 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.42 g, 1.78mmol) was added under dry nitrogen. The reaction mixture was stirred on ice for 3 hours. The solvent was evaporated and compound 3 was purified by chromatography (50:50:3 ethyl acetate:hexane / triethylamine) (yield: 70%). 1H NMR (300 MHz, CDCl3): δ 8.3-6.6 (m, 21H), 5.82 (d, 1H), 4.4-4.2 (m, 2H), 3.8 (s, 3H), 3.7 (d, 6H), 3.6-3.1 (m, 8H), 2.5 (m, 1H), 2.4-2.2 (m, 5H), 1.3-0.9 (m, 20H). 31P NMR (CDCl3) 149.

[0179] (Size Exclusion Chromatography) Size exclusion chromatography was performed on a Shimadzu HPLC system equipped with a SEC-biosil column (repacked into a 200 x 4.6 mm column). Samples were eluted with 1 x PBS + 20 mM KCl at a flow rate of 0.5 mL / min. In a typical experiment, 80 μL of 5 μM Lipo-G in 1 x PBS + 20 mM KCl was diluted with 100 μL of 100 μL of 100 μM Lipo-G in 1 x PBS + 20 mM KCl. n T 10-n CpG-Fam was added to 20 μL FBS (Greiner Bio-one), the samples were vortexed briefly and incubated at 37° C. for 2 hours, then diluted in 500 μL 1×PBS containing 20 mM KCl, and the samples were then analyzed by SEC, with FBS monitored using absorbance at 280 nm while ODN was monitored at 480 nm (Fam peak).

[0180] (Circular dichroism spectrometer measurement) 5 μM CpG ODN was dissolved in 1× PBS containing 20 mM KCl. Circular dichroism (CD) spectra were recorded on an Aviv Model 202 Circular Dichroism Spectrometer at 20° C. Scans were taken from 220 nm to 320 nm at a 100 nm / min scan speed with a 1 nm bandwidth. For each spectrum, three scans were averaged and the spectral contribution of the buffer was subtracted.

[0181] (Animals and cells) Animals were housed in a USDA-inspected MIT Animal Facility under federal, state, local and NIH guidelines for animal care. C57BL / 6 albino mice (6-8 weeks old) were obtained from the Jackson Laboratory. Cells were cultured in complete medium (MEM, 5% fetal bovine serum (Greiner Bio-one), 100 U / ml penicillin G sodium and 100 μg / ml streptomycin (Pen / Strep), MEM sodium pyruvate (1 mM), NaH2CO3, MEM vitamins, MEM non-essential amino acids (all from Invitrogen), 20 μM β-mercaptoethanol (β-ME)).

[0182] (statistical analysis) All error bars represent SEM. Comparison of mean values ​​was performed using unpaired Student's t-test. *, p<0.05; **, p<0.01; ***, p<0.001. Graphpad Prism 5 software was used.

[0183] (result) Albumin serves as the main fatty acid transporter in extracellular fluids. Experiments were designed to test whether antigens / adjuvants modified with lipophilic albumin binding domains accumulate in lymphoid organs after injection and transported with endogenous albumin via in situ complexation. To establish this strategy, a model vaccine was developed that contains a peptide antigen combined with CpG DNA, a single-stranded oligonucleotide containing an unmethylated cytosine-guanine motif that binds to Toll-like receptor 9 and functions as a potent molecular adjuvant.

[0184] To identify optimal albumin-binding domains that could be appended to either CpG or peptide antigens, a series of amphiphilic 20-base phosphorothioate (PS)-stabilized CpG oligos linked to various lipophilic tails via their 5'-ends (amph-CpGs), 3'-labeled with fluorescein amidite, were constructed (FAM, Fig. 1A), and the interaction of these amphiphiles with serum proteins was evaluated by size-exclusion chromatography (SEC, Fig. 2B). Fetal bovine serum (FBS) showed the major fraction of proteins eluting at 5.3 min (co-with serum albumin) in SEC. Diacyl lipid-conjugated CpG (lipo-CpGs) in aqueous solution eluted as micelles (3.7 min), but after 2 h incubation with 20% FBS, about 46% of this amph-CpG co-migrated with albumin (Fig. 2B). In contrast, the majority of mono-acyl-(C18-CpG) and cholesterol-(Cho-CpG) oligos eluted as unimers at 5.8 min, essentially identical to unmodified CpG in the presence or absence of serum, indicating the stability of the PS backbone against serum nuclease degradation and the lack of interaction with albumin (Figure 2B).

[0185] Spectrophotometric measurements of FRET between FAM-labeled lipo-CpG and rhodamine-conjugated albumin confirmed the molecular association of the diacyl lipid amphiphiles with albumin in solution (FIGS. 1F and 1G).

[0186] To determine whether CpGs with different affinities for albumin show differential LN targeting, amph-CpG was injected sc at the base of the tail of mice, and 24 hours later, draining inguinal and axillary LNs were excised and analyzed intact by IVIS fluorescence imaging. C18-CpG and Cho-CpG showed slightly increased uptake in LNs compared to unmodified CpG. In contrast, lipo-CpG showed a dramatic increase in LN accumulation, 8-fold higher than soluble CpG at 24 hours and much higher than CpG delivered in incomplete Freund's adjuvant or poly(ethylene glycol) (PEG)-coated liposomes, two prototype vaccine vehicles. As shown by previous studies, the PS backbone used to stabilize the CpG oligos against serum nucleases promotes nonspecific binding to the extracellular matrix at the injection site, resulting in slow clearance of the oligos from tissues over several days. However, soluble CpG levels reached an earlier, lower peak in the proximal LNs and did not accumulate above 0.3% of the injected dose at any time point (Figure 2C). In contrast, lipo-CpG was detected in LNs within 2 h of injection and continued to accumulate for 3 days before decaying, giving a higher total AUC of exposure to CpG in the draining LNs than soluble CpG over the week after injection. LN accumulation was not dependent on TLR-9-recognized CpG motifs because non-CpG polythymidine amphiphiles (lipo-T 20 ) was also detected at high levels in LNs (Figure 2J).

[0187] Example 2: Stabilized micelles demonstrate reduced lymph node targeting (material and method) (Flow Cytometry) All antibodies were purchased from BD pharmingen or ebioscience. Cells were acquired on a FACScanto flow cytometer (BD biosciences) and analyzed using flowjo software (Tree Star Inc. Ashland, OR).

[0188] (Intracellular cytokine staining (ICCS)) Cells were plated in 96-well round-bottom plates and pulsed with minimal peptide in complete medium at 37° C. for 6 hours in the presence of Brefeldin A. Cells were stained with anti-CD8-APC and then fixed using Cytofix (BD biosciences) according to the manufacturer's instructions. Cells were then washed and permeabilized. Intracellular staining with anti-INF-γ-PE and anti-TNF-α-FITC was then performed according to BD's protocol. FACS data was collected and analyzed as previously described.

[0189] (Immunohistochemical staining) Immunofluorescence staining was performed on 10 μm frozen sections of lymph node biopsy specimens. To reduce FITC bleaching, sections were mounted with Vectashield mounting medium (Vector Laboratories, Inc. Brulingame, CA) and viewed under a Zeiss LSM 510 microscope (Oberkochen, Germany). Staining on lymph node sections was performed directly with PE-labeled CD11c and APC-labeled F4 / 80, or PE-labeled B220 and APC-labeled CD3 antibodies.

[0190] (result) The in vitro analysis of Example 1 shows that lipo-CpG molecules achieve a parallel transition between micellar and albumin-bound forms in the presence of serum. However, the enhanced lymph node accumulation achieved by these amphiphiles could be driven by either species. To distinguish between these possibilities, polyguanine repeats were introduced between the diacyl lipid and the CpG sequence. Lipo-G containing four or more guanine repeats were n G-quadruplex hydrogen bonds between adjacent oligo strands in -CpG micelles blocked albumin access to the lipid tails, stabilizing the micelles against degradation in the presence of serum (discussed in more detail below).

[0191] While albumin-conjugated lipo-CpG and lipo-G2-CpG showed strong LN targeting, G-quartet-stabilized lipo-G4-CpG or lipo-G6-CpG micelles showed very poor LN accumulation after sc injection (Figure 8A). (Note that the effect of different oligo lengths is negligible here, since lipo-T6-CpG showed similar LN accumulation.) Longitudinal analysis of CpG fluorescence at the injection site and in the draining LNs indicated that lipo-G 4 / 6 It was shown that the low LN accumulation of -CpG amphiphiles was due to the lack of flux of the stabilized micelles from the injection site. Amplification of nonspecific matrix binding by the PS DNA backbone in multivalent micellar forms may irreversibly trap a large proportion of stabilized micelles at the injection site.

[0192] Consistent with the IVIS data, little detectable accumulation of CpG or lipo-G4-CpG was observed in tissue sections of the draining inguinal LNs, whereas lipo-CpG and lipo-G2-CpG accumulated in the subcapsular sinus and in interfollicular areas extending towards the paracortex. Immunohistochemical and flow cytometric analyses demonstrated that these LN-accumulating amphiphiles were expressed in the F4 / 80 + Macrophages and CD11c + We showed that it colocalized with dendritic cells (Fig. 3E).

[0193] Example 3: Albumin "hitchhiking" targets lipo-oligo conjugates to lymph nodes (material and method) (Albumin-CpG conjugate) Mouse serum albumin (10 mg in 200 μL PBS) was added to 0.79 mg BMPS (Aldrich) dissolved in 20 μL DMSO. The mixture was stirred at room temperature for 2 hours. Excess BMPS was removed by passing the mixture through a G-25 column. Then, 246 μg disulfide-labeled fluorescein-CpG (preactivated with 20 μL 100 mM TCEP) was added to the solution. The mixture was reacted overnight to dialyze excess CpG (50K MWCO) and the absence of free CpG was confirmed by size exclusion chromatography.

[0194] (result) If albumin "hitchhiking" is required to optimally target CpG molecules to LNs, covalent conjugation of oligos to albumin should result in similar LN accumulation. To test this, CpG was covalently conjugated to mouse serum albumin (MSA) and the LN uptake of these conjugates was compared to lipo-CpG or soluble CpG. A statistically significant difference was observed between the conjugates and lipo-ODN, and the fluorescence intensity of both MSA-CpG and lipo-CpG in LNs was much greater than that of soluble ODN. Overall, these data indicate that efficient LN accumulation of CpG oligonucleotides conjugated to lipophilic tails depends on the ability of the amphiphile to partition from micelles into a serum protein-bound state.

[0195] Example 4: Albumin-bound lipo-oligo conjugates enhance immune responses while minimizing systemic toxicity in vivo. To determine the effect of CpG targeting to LNs on the immune response, mice were cultured with unmodified CpG, CpG in IFA, albumin-bound CpG (Lipo-G n -CpG, n=0,2) or G-quadruplex-stabilized CpG micelles (Lipo-G nAnimals were primed on day 0, boosted on day 14, and CD8+ T cell responses were analyzed on day 20. Administration of lipid-conjugated CpG, but not unconjugated CpG (soluble CpG or CpG emulsified in IFA), resulted in a significant increase in the CD8+ T cell response compared to unmodified CpG alone or CpG emulsified in IFA, compared to OVA. 257-264 CD8 specific for + The strongest responses were elicited by albumin-bound lipo-CpG and lipo-G2-CpG (Figure 4A).

[0196] Intracellular cytokine staining in peripheral blood lymphocytes showed qualitatively the same trend, with the frequencies of IFN-g- and TNF-a-producing T cells being greatly expanded by the albumin-conjugated CpG amphiphiles (Fig. 4B). Control immunizations with the non-TLR agonist Lipo-GpC or with the PEG conjugate Lipo-(PEG) with 48 ethylene glycol units mixed with OVA elicited minimal responses, except for the direct adjuvant diacyl lipid tail.

[0197] Repeated subcutaneous injections of high-dose albumin-bound CpG did not induce systemic nonspecific immune activation in vivo, as characterized by systemic proinflammatory cytokine release (Figure 10) and lymphocyte activation in the spleen (splenomegaly, Figure 4D). In contrast, administration of free CpG in mice resulted in systemic toxicity (Figures 10, 11). Taken together, these experiments strongly suggest that lymph node-targeted amph-CpG is a potent adjuvant that can induce large-scale CD8 T cell responses while avoiding systemic immune activation.

[0198] Example 5: Albumin "hitchhiking" targets lipo-peptide conjugates to lymph nodes (material and method) (Synthesis of Fluorescein-PEG Amphiphiles) PE lipids (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine, DMPE; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPE; 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, DPPE; 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine, DSPE, Avanti polar lipids. Inc.) were dissolved in 500 μL CHCl3 and 500 μL DMF and 1.2 equivalents of fluorescein-PEG 2000 -NHS (creative PEG works Inc.) was added, the reaction mixture was stirred overnight, and the amphiphilic fluorescein PEG amphiphile was purified by reverse-phase HPLC using a C4 column (BioBasic-4, 200 mm x 4.6 mm, Thermo Scientific) and 100 mM triethylamine-acetate buffer (TEAA, pH 7.5)-methanol (0-30 min, 10-100%) as eluent.

[0199] (Synthesis of peptide amphiphiles) The N-terminal cysteine ​​modified peptide was dissolved in DMF and 2 equivalents of maleimide-PEG 2000 -DSPE (Laysan Bio, Inc.) and the mixture was stirred at room temperature for 24 hours. Bioconjugation was determined to be essentially complete by HPLC analysis. The peptide conjugate was then diluted in 10x ddH2O, lyophilized to a powder, redissolved in H2O, and stored at -80°C.

[0200] (result) The synthesis of lipo-CpG is straightforward as solubility is facilitated by the long polar oligonucleotide block, but depending on the amino acid sequence, lipidated peptides can be essentially insoluble. Thus, to generalize the lymph node targeting approach achieved with lipo-CpG to peptide antigens and other potential vaccine components, lipo-PEG amphiphiles composed of diacyl lipid tails (amph-peptides; e.g., Figure 1C) linked to peptide cargoes via polar PEG blocks were generated using ethylene glycol spacers of various lengths to mimic the long polar blocks of lipo-CpG.

[0201] The length of the PEG block in this design controls the balance of the three-way equilibrium in physiological conditions: amph-peptides and lipo-PEG in pure water form micelles, whereas in the presence of serum and cells, these amphiphiles reach equilibrium between binding to albumin and intercalating their diacyl tails into the cell membrane (Figure 2F). Lipo-PEG-FAM amphiphiles with short PEG blocks showed stable plasma membrane intercalation when incubated with cells in the presence of serum in vitro (Figure 2G), which blocked migration into LNs relative to albumin in vivo. However, increasing the polar block to 48 ethylene glycol units gave lipo-PEG amphiphiles that partitioned into solution while retaining albumin binding (Figure 2G). From this in vitro behavior, the influx pattern in vivo was directly extrapolated. This is because sc injected lipo-PEG-FAM amphiphiles showed increased LN accumulation with increasing PEG block length (48 EG units compared to 4 EG units, Figure 2H).

[0202] A similar trend was observed for DNA amphiphiles; lipo-Tn oligos prepared with increasing polythymidine chain length showed increasing accumulation in LNs after sc injection until accumulation plateaued for the oligos (Figure 2J). As with CpG amphiphiles, the structure of the hydrophobic block was also important; lipo-PEG amphiphiles with long diacyl tails (>16 carbons, which show high affinity for albumin) showed strong fluorescence in lymph nodes, while shorter lipid tails with poor affinity for albumin showed low LN accumulation (Figure 2I).

[0203] Example 6: Lymph node targeted vaccine induces immune response (material and method) (Vaccine components) Minimal peptide was purchased from Anaspec; ovalbumin was purchased from Worthington Biochemical Corporation; Cysteine ​​(Cys) modified peptide HPV-16 E7 49-57 (CRAHYNIVTF), AL-11 (CAAVKNWMTQTL) and Trp-2 (CSVYDFFVWL) were synthesized by GenScript and purified by reversed-phase HPLC. 2000 -Maleimide was purchased from Laysan Bio Inc. CpG ODN was synthesized in-house. IFA was purchased from Sigma-Aldrich.

[0204] (Vaccine preparation) Mice were vaccinated with a prime-boost regimen. Typically, each prime and boost vaccine in the experiment consisted of the following components: 10 μg OVA, 20 mM K + , 10mM Mg + 1.24 nmol CpG suspended in 1x PBS containing 0.1% ethanol. In experiments using IFA, CpG / OVA was combined with an equal volume of IFA and emulsified thoroughly. The volume of all vaccine injections was 100 μl. For peptide micelles, mice were immunized with 20 mM K +, 10mM Mg + 10 μg of peptide-PEG mixed with 1.24 nmol CpG suspended in 1x PBS containing 2000 20 μg of peptide-PEG primed with -DSPE conjugate and mixed with 1.24 nmol CpG 2000 Mice were boosted with -DSPE conjugate by sc injection at the base of the tail.

[0205] (Tetramer staining) Tissue samples were collected and single cell suspensions (spleen and lymph nodes) were prepared. Blood was collected and red blood cells were depleted with ACK lysis buffer. Cells were then blocked with Fc-blocker (anti-mouse CD16 / CD32 monoclonal antibody) and stained with PE-labeled tetramer (Beckman Coulter) and anti-CD8-APC for 30 minutes at room temperature. Cells were washed twice and resuspended in FACS buffer. FACS data were collected on a BD FACScanto flow cytometer and analyzed using flowjo software. Analysis typically included CD8 + , gated on tetramer-positive live cells.

[0206] In vivo cytotoxicity assay Splenocytes from naive mice were pulsed or not with 10 μM SIINFEKL peptide for 30 min, then cells were labeled with either 1 μM (for pulsed cells) or 0.1 μM (for control cells) CFSE for 10 min at 37° C. and washed extensively. Cells were mixed in a 1:1 ratio and diluted with 10×10 6 Total cells were injected iv into mice pre-challenged with the vaccine formulation as described above. Eighteen hours later, splenocytes from each recipient mouse were analyzed by FACS to detect CFSE-labeled cells.

[0207] (result) Based on the design rules for efficient targeting of compounds to lymph nodes discussed above, peptide antigens were grafted onto commercially available DSPE-PEG (a diacyl lipid tail with 18 carbons, 2KDa PEG block) to generate amph-peptides for use in vaccination studies (Figure 9).

[0208] Having established the structure-function relationship between albumin binding and lymph node retention, experiments were designed to test whether combining antigens with CpG amphiphiles could directly prime an antigen-specific immune response. Various peptide antigens, including viral antigens (SIV gag, AL11), tumor associated self-antigens (melanoma antigen, Trp2), and tumor specific antigens (human papillomavirus, type 16, E7, HPV-16-E7), were coupled to maleimide-functionalized DSPE-PEG 2000 Antigen conjugation did not significantly affect albumin binding.

[0209] After vaccination, the induced CD8 T cell responses and functionality were monitored using tetramer techniques or intracellular cytokine staining (ICS) as discussed above. 2000 Administration of a vaccine consisting of a 100-mg IgG1-peptide adjuvant (Lipo-G2-CpG) dramatically increased antigen-specific CD8 expression against all of the minimal peptide epitopes listed above. + Mice vaccinated with amph-Trp2+amph-CpG generated CD8 T cell responses (Figures 5A-5B). + An average of 15% and 7% of lymphocytes produced IFN-γ and TNF-α, respectively. In contrast, the two control groups that received free Trp2 showed only negligible CTL activity (FIG. 5B).

[0210] Direct conjugation of lipids to antigens without a PEG linker resulted in a dramatic reduction in immune responses, indicating that a long PEG linker is required to elicit CD8 T cell immune responses (Figure 5C). This observation was consistent with previously observed LN accumulation data, in which efficient LN retention required a long PEG spacer. The above data indicate that albumin-conjugated vaccine formulations elicit large numbers of functional antigen-specific CD8 + It was consistently observed that mice immunized with the self-delivered formulation had more potent cytotoxic activity against peptide-pulsed target populations compared to the unpulsed controls (Figure 5D).

[0211] Example 7: Lymph node targeted vaccine shows therapeutic efficacy The therapeutic benefit of the CD8 response generated after immunization was tested by treating established subcutaneous mouse tumors, TC-1, which express the E7 oncoprotein derived from human papillomavirus type-16 (HPV-16). Six to eight week old C57BL / 6 mice were inoculated with TC-1 tumor cells (3 × 10 5 Mice were inoculated subcutaneously into the upper left flank with 100 μg / mouse (100 μg / mouse). After palpable tumors formed (day 6), mice were randomized and administered unconjugated E7 49-57 Using peptide and CpG as controls, mice were injected with amph-HPV (DSPE-PEG-E7) in combination with amph-CpG (Lipo-G2-CpG) at the base of the tail. 49-57 ) and tumor growth was monitored every 2-3 days.

[0212] As shown in Figure 5E and Figure 5F, tumors grew rapidly in unvaccinated mice. Mice treated with vaccine amphiphiles inhibited the growth of subcutaneously growing TC-1 tumors (3-5 mm in diameter at first treatment) for several weeks. In contrast, treatment with unconjugated CpG oligonucleotides + HPV-16 E7 peptide antigen had only a modest antitumor effect (Figure 5E and 5F), resulting in a temporary delay in tumor growth by day 19, after which tumors progressed rapidly. Taken together, the above results demonstrate that the combination of amph-peptide antigen with amph-CpG adjuvant dramatically enhances antigen-specific CTL responses, resulting in improved antitumor immunity in mouse tumor models.

[0213] Example 8: G-quadruplex linkers stabilize oligonucleotide micelles (material and method) (Oligonucleotide synthesis) Oligonucleotides were synthesized on an automated DNA synthesizer (ABI 394, Applied Biosystems, Inc.) at a 1.0 micromolar scale. All DNA synthesis reagents, including cholesteryl-triethylene glycol (TEG)-phosphoramidites and DMT-polyethylene glycol (PEG)-phosphoramidites, were purchased from Glenres and Chemgenes and used according to the manufacturer's instructions. The immunostimulatory cytosine-guanine (CG) oligonucleotide was a type B sequence designated 1826 (Lipo-G n -CG: 5'-diacyl lipid-G n-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 1). Synthesis and solid phase conjugation of lipid phosphoramidites followed previous reports. Particle size was determined by dynamic light scattering (DLS) using a 90Plus / ZetaPals particle size and ξ potential analyzer (Brookhaven Instruments). DSPE-PEG2000-maleimide was purchased from Laysan Bio Inc. Carboxyfluorescein-labeled PEG2000-DSPE was purchased from Avanti Polar lipids Inc.

[0214] (Circular dichroism) 5 μM of CG oligonucleotide was dissolved in 1× phosphate-buffered saline (PBS) containing 20 mM KCl. Circular dichroism (CD) spectra were measured using an Aviv Model Recordings were made on a 202 Circular Dichroism Spectrometer at 20° C. Scans were taken from 220 nm to 320 nm at a scan speed of 100 nm / min with a 1 nm bandwidth. For each spectrum, an average of three scans was taken and the spectral contribution of the buffer was subtracted.

[0215] (Size Exclusion Chromatography) Size exclusion chromatography was performed on a Shimadzu HPLC system equipped with a SEC-biosil column (repacked into a 200 x 4.6 mm column). Samples were eluted with 1x PBS + 20 mM KCl at a flow rate of 0.5 mL / min. In a representative experiment, fluorescein-labeled DNA micelles (5 μM Lipo-G in 1x PBS + 20 mM KCl) were used. n T 10-nCG-Fam (80 μL) was incubated with 20% fetal bovine serum (FBS) (20 μL) (Greiner Bio-one), the samples were vortexed briefly, incubated at 37° C. for 2 hours, and then diluted with 500 μL 1×PBS+20 mM KCl. Samples were then analyzed by SEC. Fetal bovine serum (FBS) was monitored using absorbance at 280 nm, while oligonucleotides were monitored at 480 nm (Fam peak).

[0216] (result) Guanine (G)-rich nucleic acid sequences can fold into various types of G-quadruplex structures (Davis, JT Angew. Chem. Int. Ed. Engl. 43, 668-698 (2004)) (e.g., intramolecular, intermolecular, parallel, and antiparallel). To promote micellar self-assembly and minimize oligonucleotide folding, the lipid-oligonucleotide conjugates were first suspended in pure water and then diluted with potassium (K + )-containing buffer was added to stabilize the G-quadruplex. Micelle formation was confirmed by transmission electron microscopy, dynamic light scattering measurements, and size-exclusion chromatography. Figure 7B illustrates the size profile of the self-assembled micelles, which show a homogenous size distribution.

[0217] Circular dichroism (CD) was performed to characterize the formation of G-quadruplexes. Lipo-G0T 10 The spectrum of -CG oligonucleotides showed a small negative peak around 245 nm and a positive peak around 278 nm, while changing the number of guanines from 0 to 10 induced parallel G-quadruplexes, as revealed by a shift of the positive peak from 278 nm towards 262 nm (the characteristic band of a parallel G-quadruplex), while retaining the negative 245 nm band (Paramasivan, S., et al., Methods 43, 324-331 (2007)) (Figure 7C).

[0218] The design of G-quadruplex-stabilized CpG adjuvants is shown in Figure 3A. G-quadruplex-stabilized CpG micelles are self-assembled from three distinct segments: immunostimulatory CpG-ODN, an ODN composed of a central repeat block containing G-quartet-forming guanines, n=1-10, followed by 10-n non-interacting thymidines, and a diacyl lipid tail (Figure 3A). Pyrene excimer fluorescence was used to assay the stability of G-quadruplex micelles in the presence of albumin (Figure 3B). Pyrene dyes (n>2) incorporated into stabilized CpG micelles retain excimer fluorescence in the presence of high concentrations of albumin. In contrast, albumin binds to the lipid moiety of non-stabilized micelles (n≦2), disrupting the micellar structure and resulting in a decrease in excimer fluorescence in an albumin concentration-dependent manner (Figure 3B). The stability of the DNA micelles in the presence of serum proteins was also investigated by size-exclusion chromatography (SEC) (Figure 3C). Since the micelles have a relatively high molecular weight, they eluted at 3.7 min, while FBS showed a major peak at 5.3 min. After incubation, the non-stabilized micelles (Lipo-G0T 10 In the presence of guanine, 20% of the micelles (lipo-G2T8-CG) remained intact, whereas the remaining 80% were destroyed and bound to the FBS components (peaking at 5.2 min). In the presence of guanine, the percentage of intact micelles increased from 36% (lipo-G2T8-CG) to 73% (lipo-G4T6-CG) and finally reached 90% (lipo-G6T4-CG). By increasing the number of guanines to 8 (lipo-G8T2-CG) and 10 (lipo-G 10 Increasing the number of guanines between the CPG-oligonucleotide and lipid tail controls the micelle stability in the presence of serum proteins, as evidenced by the FBS peak.

[0219] Taken together, these experiments demonstrated that the G-quadruplex micelle stability under micelle-disrupting conditions can be controlled by varying the number of guanines.

[0220] Example 9: G-quadruplex linkers affect lymph node accumulation and cellular uptake (material and method) (mouse) C57BL / 6 albino mice (6-8 weeks old) were obtained from Jackson Laboratory. Animals were maintained at the USDA-inspected Massachusetts Institute of Technology under federal, state, local and NIH guidelines for animal care. The animals were housed at the MIT Animal Facility.

[0221] (Isolation of bone marrow cells) Bone marrow-derived dendritic cells were prepared according to a modification of the Inaba procedure, as previously reported. Dendritic cells were activated / matured with 500 nM CG probe for 12 h and washed three times with PBS before use. Cells were cultured in complete medium (MEM, 5% fetal bovine serum (Greiner Bio-one), 100 units (U) / ml penicillin G sodium and 100 μg / ml streptomycin (Pen / Strep), MEM sodium pyruvate (1 mM), NaH2CO3, MEM vitamins, MEM non-essential amino acids (all from Invitrogen), and 20 μM β-mercaptoethanol (β-ME)).

[0222] In vivo imaging and flow cytometry The draining lymph nodes of each group of mice were analyzed 24 and 72 hours after injection by In Vivo Imaging Systems (IVIS®) and flow cytometry. All antibodies for flow cytometry were purchased from BD Pharmingen or Ebioscience.

[0223] (statistical analysis) All error bars represent SEM. Comparison of means was performed using unpaired Student's t-test. *, p<0.05; **, p<0.01; ***, p<0.001. GraphPad Prism 5 software was used.

[0224] (result) The lymphatic system absorbs interstitial fluid from tissues and returns it to the blood via lymph nodes. Animal studies were performed to evaluate micelle targeting to the lymphatic system. Dye-labeled CG oligonucleotides, dye-labeled CG oligonucleotides emulsified in IFA, or dye-labeled Lipo-G were administered. n -CG micelles (n=0, 2, 4 or 6) were subcutaneously injected into separate groups of mice. The draining lymph nodes of each group of mice were analyzed 24 and 72 hours after injection by In Vivo Imaging Systems (IVIS®) and flow cytometry. Cells were acquired on a FACScanto flow cytometer (BD Biosciences) and analyzed using Flowjo software (Tree Star Inc. Ashland, OR).

[0225] All lymph nodes became visibly larger and reached maximum enlargement at 24 hours. Fluorescence imaging of isolated lymph nodes at 24 and 72 hours revealed significant differences between the different mouse groups. Moderately stable Lipo-G was retained by the inguinal lymph nodes (proximal lymph nodes) and axillary lymph nodes (distal lymph nodes). n The number of CpG micelles (n = 0 or 2) was determined by the amount of retained excess stabilized lipo-G. n Peak lymph node targeting was achieved by lipid-G2-CpG micelles, which was greater than that achieved by lipid-G2-CpG micelles (n=4 or 6) (Figures 8A and 8B). Seventy-two hours after injection, labile lipid-G (0もしくは2) Uptake of Lipo-CpG micelles was increased 5-fold, uptake by macrophages was increased 8-fold, and uptake by B cells was increased 5-fold compared to soluble CpG oligonucleotides. In contrast, the more stable Lipo-G (4もしくは6) -CG micelles showed low levels of lymph node retention and cell association.

[0226] Example 10: Immunostimulatory micelles stimulate antigen-specific CD8 + Induces T cell proliferation) (material and method) Mouse CD8 + T cell proliferation was moderately stable using soluble CpG oligonucleotides as a control (Lipo-G (0もしくは2) -CpG oligo) or overstable (Lipo-G (4もしくは6) The study was conducted after immunization / vaccination with 100 mM K-CpG oligo (immunostimulatory micelle) mice. C57Bl6 (B6) mice were vaccinated on days 0 and 14 and analyzed on days 20 or 21. Typically, each injection contained the following components: 10 μg ovalbumin (OVA) antigen (purchased from Worthington Biochemical Corporation) and 1× PBS (20 mM K + and 10 mM Mg + 1.24 nmol Lipo-G suspended in n -CG micelles. Ovalbumin (OVA) was used as a model antigen because it has a well-studied H-2 Kb-restricted MHC class I epitope in B6 mice. In experiments using Freund's incomplete adjuvant (IFA), a volume of soluble CpG oligonucleotide and soluble OVA antigen were combined and emulsified with an equal volume of IFA. The total volume of each vaccine injection was 100 μl. Mice were injected subcutaneously at the base of the tail. After immunization, blood samples were collected from the spleen and lymph nodes, and single cell suspensions were prepared (red blood cells were depleted by ACK lysis buffer). The blood sample preparations were then used to infect SIINFEKL-specific CD8 + MHC class I-restricted OVA to track T cell proliferation 257-264 The results were evaluated by tetramer staining. Cys-modified peptide OVA 257-264(CSIINFEKL) was synthesized by GenScript and purified by reversed-phase HPLC. Cells were then blocked with Fc-blocker (anti-mouse CD16 / CD32 monoclonal antibody) and stained with PE-labeled Kb / SIINFEKL tetramer (Beckman Coulter) and anti-CD8-APC for 30 min at room temperature. Cells were washed twice and resuspended in FACS buffer. FACS data were collected on a BD FACScanto flow cytometer and analyzed using Flowjo software. Analysis typically included CD8 + , gated on tetramer-positive live cells.

[0227] (result) The administration of the immunostimulatory micelles was 257-264 Unexpectedly, moderately stabilized Lipo-G2-CpG oligo-based micelles induced proliferation of SIINFEKL-specific CD8+ T cells (Figures 4A and 4B). + Six days after the second (boost) injection of the labile Lipo-G2-CpG oligo-based micelles, approximately 33% of all CD8+ T cells detected in the blood were specific for the antigen, whereas 10% of all CD8+ Only about 7% of T cells were antigen-specific after boosting with stabilized Lipo-G6-CpG oligo-based micelles, and thus the strength of the T cell response stimulated by this vaccine directly correlated with the moderately stabilized CpG micelles showing the greatest accumulation in lymph nodes.

[0228] CD8 +To test the responsiveness of T cells, blood lymphocytes were restimulated ex vivo with OVA-specific peptide SIINFEKL for 6 hours and analyzed for the production of cytokines IFN-γ and TNF-α. Cells were plated in 96-well round-bottom plates and pulsed with minimal peptide in the presence of brefeldin A for 6 hours at 37°C in complete medium. Cells were stained with anti-CD8-APC and then fixed using Cytofix (BD biosciences) according to the manufacturer's instructions. Cells were then washed and permeabilized. Intracellular staining for anti-INF-γ-PE and anti-TNF-α-FITC was then performed according to BD's protocol. FACS data were collected and analyzed as described. Again, the destabilized Lipo-G2-CpG oligo-based micelles were the most effective, correlating with the above findings (Figure 4B).

[0229] Further in vivo cytotoxic lymphocyte (CTL) assays were performed to determine the extent of the expanded CD8 + To assess whether the T cell population was functional, splenocytes from naive mice were pulsed or not with 10 μM SIINFEKL peptide for 30 min. The cells were then labeled with either 1 μM (for pulsed cells) or 0.1 μM (control cells) CFSE for 10 min at 37° C. and washed extensively. The cells were mixed in a 1:1 ratio and diluted with 10×10 6 Total cells were injected intravenously (iv) into mice pre-challenged with the vaccine formulation as described above. After 18 hours, splenocytes from each recipient mouse were analyzed by FACS to detect CFSE-labeled cells. n CD8 in mice immunized with -CG-based micelles + T cells lysed >97.9% of the peptide-pulsed target population, whereas CD8 T cells lysed >97.9% of the peptide-pulsed target population from mice immunized with soluble CpG oligonucleotides. + T cells lysed an average of 54.6% of target cells.

Claims

1. 1. An amphiphilic albumin binding conjugate comprising: (a) a lipid component; (b) any polar component; and (c) an immunomodulatory compound or molecular adjuvant; Including, wherein the immunomodulatory compound or molecular adjuvant is directly attached to the lipid or is attached to the lipid via a linker; the conjugate is sufficiently soluble such that the lipid binds to albumin under physiological conditions, and A number of said conjugates are capable of spontaneously forming micelles in aqueous solution. Amphiphilic albumin binding conjugates.

2. The conjugate of claim 1 , wherein the immunomodulatory compound or molecular adjuvant is attached to the lipid via a linker.

3. The conjugate of claim 2 , wherein the linker is an oligonucleotide linker.

4. The conjugate of claim 3, wherein the oligonucleotide linker comprises "N" consecutive guanines, where N is between 0 and 2.

5. Structure L-5'-G ｎ 5. The conjugate of claim 4, comprising the amino acid sequence: -ON-3', where "L" is the lipid, "G" is guanine, "n" is 0-2, and "ON" is the immunostimulatory oligonucleotide.

6. The conjugate of any one of claims 1 to 5, wherein the oligonucleotide conjugate, when administered to a subject in vivo, exhibits increased accumulation in the lymph nodes compared to administration of the immunostimulatory oligonucleotide alone.

7. The conjugate according to any one of claims 1 to 6, wherein the lipid is a diacyl lipid.

8. The conjugate according to any one of claims 1 to 7, wherein the acyl chain of the lipid comprises from 12 to 30 carbohydrate units.

9. The conjugate of any one of claims 1 to 8, wherein the immunostimulatory oligonucleotide is capable of binding to a pattern recognition receptor.

10. The conjugate of claim 9 , wherein the immunostimulatory oligonucleotide comprises CpG.

11. The conjugate of claim 10, wherein the immunostimulatory oligonucleotide is a ligand for a Toll-like receptor.

12. The conjugate of any one of claims 1 to 11, wherein the immunostimulatory oligonucleotide has a phosphorothioate (PS) backbone.

13. The conjugate according to any one of claims 1 to 12, wherein the oligonucleotide comprises 20 or more nucleic acids.

14. A vaccine adjuvant comprising a plurality of oligonucleotide conjugates according to claims 1 to 13. to.

15. 1. An oligonucleotide conjugate comprising an immunostimulatory oligonucleotide linked to a linker comprising at least three consecutive guanines conjugated to a lipid, wherein a plurality of said oligonucleotide conjugates are capable of spontaneously forming micelles in aqueous solution; and More than 36% of the micelles are intact in the presence of 20% fetal bovine serum. Oligonucleotide conjugates.

16. The oligonucleotide conjugate has the structure L-5'-G ｎ 16. The oligonucleotide conjugate of claim 15, comprising the amino acid sequence: -ON-3', where "L" is the lipid, "G" is guanine, "n" is 3 to 10, and "ON" is the immunostimulatory oligonucleotide.

17. 17. A vaccine adjuvant comprising a plurality of oligonucleotide conjugates according to any one of claims 15 or 16.

18. An amphipathic peptide conjugate comprising a peptide antigen, the peptide antigen comprising: (i) directly conjugated to a lipid, or (ii) linked to a linker that is conjugated to a lipid; wherein the lipid binds to albumin under physiological conditions, the peptide antigen, the linker, or the peptide antigen and linker in combination, are sufficiently polar to reduce or inhibit entry of the lipid into the plasma membrane of a cell; Amphipathic peptide conjugates.

19. 20. The peptide conjugate of claim 18, wherein the peptide antigen is linked to a linker that is conjugated to the lipid.

20. 20. The peptide conjugate of claim 19, wherein the linker is selected from the group consisting of a hydrophilic polymer, a hydrophilic amino acid chain, a polysaccharide, or a combination thereof.

21. 20. The peptide conjugate of claim 19, wherein the linker comprises "N" consecutive polyethylene glycol units, where N is between 25 and 50.

22. The peptide conjugate of any one of claims 18 to 21, wherein the peptide conjugate, when administered to a subject in vivo, exhibits increased accumulation in the lymph nodes compared to administration of the antigenic peptide alone.

23. The peptide conjugate according to any one of claims 18 to 22, wherein the lipid is a diacyl lipid.

24. The peptide conjugate according to any one of claims 18 to 23, wherein the acyl chain of the lipid comprises from 12 to 30 carbohydrate units.

25. An immunogenic composition comprising the adjuvant of claim 14 and an antigen.

26. The immunogenic composition according to claim 25, wherein the antigen is a peptide conjugate according to any one of claims 18 to 25.

27. 20. An immunogenic composition comprising the adjuvant of claim 17 and an antigen.

28. A method for increasing an immune response in a subject, the method comprising the step of administering to the subject an effective amount of an immunogenic composition according to any one of claims 25 to 27, thereby increasing the immune response in the subject.

29. 29. The method of claim 28, wherein the immune response is an increase in the number of CD8+ T cells expressing TNF or INF compared to a control.

30. The method of claims 28-29, wherein the subject has cancer or an infectious disease.

31. A method for treating cancer or an infectious disease, the method comprising the step of administering to a subject an effective amount of an immunogenic composition according to any one of claims 25 to 27, to reduce one or more symptoms of the cancer or infectious disease compared to a control.

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