KR101986407B1 - Adjuvant composition comprising ascophyllan as active ingredient - Google Patents

Abstract

The present invention relates to an adjuvant composition comprising ascorbyllan as an active ingredient and a vaccine composition comprising the adjuvant composition and a target antigen as an active ingredient, Induces maturation of dendritic cells, increases immune response and CTL activity of Th1 cell mediated by matured dendritic cells, and induces an antigen-specific immune response when treating ascorbicin and target antigen together to prevent cancer Or treatment, as well as inhibiting recurrence or metastasis of cancer.

Description

[0001] The present invention relates to an adjuvant composition comprising ascorbyl as an active ingredient,

The present invention relates to an adjuvant composition comprising ascopyran as an active ingredient, and a vaccine composition comprising said composition and a target antigen.

Tumor immunotherapy induces an immune response leading to the death of tumor cells (Finn OJ. The New England Journal of Medicine. 2008; 358 (25): 2704-2715). In order to effectively kill tumor cells, it is necessary to induce T cell and B cell immune responses. In particular, CTL (cytotoxic T lymphocyte) activity against tumor antigens and antigen-specific antibody production are important (Banchereau J and Palucka AK. Immunology. 2005; 5 (4): 296-306). However, tumor antigens are immune to T cell and B cell activity, suggesting that antigen presenting cells, such as DCs (dendritic cells) and macrophages, present a small amount of tumor antigen as MHC molecules, (Smith ME et al., Proceedings of the National Academy of Sciences of America, 1989; 86 (14): 5557-5561; and Jager D, Jager E and Knuth A. Journal of Clinical Pathology. 2001; 54 (9): 669-674). Thus, in order to enhance tumor immunity, adjuvants capable of inducing DC maturation are required along with tumor antigens for T cell and B cell activity (Banchereau J and Palucka AK. Nature Reviews Immunology. 2005; 5 (4): 296-306).

DC is an important regulator of T cell and B cell immunity. When exposed to stimuli or antigens, immature DCs migrate to the spleen and lymph nodes (LN) through maturation and induce T cell activity (Banchereau J and Palucka AK, Nature Reviews Immunology. ), 293-306; and Banchereau J and Steinman RM. Nature. 1998; 392 (6673): 245-252), matured DCs are determined to induce specific types of effector T cells and CTLs to induce pro-inflammatory cytokines (Moser M and Murphy KM, Nature Immunology 2000; 1 (3): 199-205; and Pooley JL, Heath WR and Shortman K. Journal of Immunology 2001; 166 (9): 5327- 5330). Other DC subsets play a different role: the minor group of mouse splenic DCs, CD8α ⁺ cDC (conventional DC), is capable of cross-presentation of intrinsically synthesized proteins and is a major member of mouse spleen DCs, CD8α ^- cDC Has the selective ability to directly direct extracellular antigens to CD4 T cells (Pooley JL, Heath WR and Shortman K. Journal of Immunology 2001; 166 (9): 5327-5330; Jin JO, Zhang W, Du JY, Wong KW, Oda T and Yu Q. PloS one. 2014; 9 (6): e99396; Schnorrer P et al., Proceedings of the National Academy of Sciences of America, 2006; 103 (28): 10729- 10734; and Watts C. Nature Immunology. 2004; 5 (7): 685-692). Because the tumor vaccine should induce CTL activity specific for tumor antigens for tumor cell killing activity, the cross-presentation of CD8α ⁺ DC-mediated to tumor antigens is very important and limited CTL because the active, mature CD8α ^- is induced by CD4 T cells, DC activation is required (Schoenberger SP, Toes RE, van der Voort EI, Offringa R and Melief CJ Nature 1998; 393 (6684): 480-483..) .

Immunoadjuvants or immunoadjuvants induce immunological activity against antigens, some of which are known as natural polysaccharides, bacterial products and cytokines (Afonso LC et al., Science. 1994; 263 (5144): 235 And Zhang W, Cho (eds.), Scientific reports, 2015, 5: 11062, 1983, pp. 273-296; McSorley SJ, Ehst BD, Yu Y and Gewirtz AT. Journal of Immunology 2002; 169 (7): 3914-3919; SY, Xiang G, Min KJ, Yu Q and Jin JO, PloS One, 2015; 10 (6): e0130926). The adjuvant function of seaweed polysaccharides appears to be important for tumor immunotherapy by inducing targeted tumor immunity (Luo M et al., Scientific Reports. 2015; 5: 11062).

Ascopyllan is a brown algae, Ascophyllum (Nakayasu S, Soegima R, Yamaguchi K and Oda T. Bioscience, Biotechnology, and Biochemistry. 2009; 73 (4): 961-964), which is a sulfated polysaccharide extracted from nodosum . Previous studies have shown that ascopyran promotes the activity of macrophages, NK cells and spleen DCs (Nakayasu S, Soegima R, Yamaguchi K and Oda T. Bioscience, Biotechnology, and Biochemistry 2009; 73 (4): 76 (8): 1573-1576; and Zhang W, Du JY, < RTI ID = 0.0 > Jiang Z, Okimura T, Oda T, Yu Q and Jin JO, Marine Drugs 2014, 12 (7): 4148-4164). However, the effect of ascorbyl on immune cell activity in an in vivo tumor environment was not investigated.

1. Japanese Laid-Open Patent Application No. 2008-120707 2. Korean Patent Publication No. 2016-0099780

1. Zhang W, Du JY, Jiang Z, Okimura T, Room T, Yu Q and Jin JO. Marine Drugs. 2014; 12 (7): 4148-4164.

It is an object of the present invention to provide an adjuvant composition comprising ascopyran as an active ingredient.

It is another object of the present invention to provide a vaccine composition comprising the adjuvant composition and the target antigen as an active ingredient.

In order to achieve the above object, the present invention provides an adjuvant composition comprising ascorbyllan as an active ingredient.

In one embodiment of the present invention, the ascorbic acid may be isolated from Ascophyllum nodosum .

In one embodiment of the invention, the ascorbic acid may induce maturation of dendritic cells (DCs) in the spleen and lymph nodes.

In one embodiment of the present invention, the ascorbyl may promote the immune response of Th1 cell mediated.

In one embodiment of the present invention, the ascopyran may increase the activity of CTL (Cytotoxic T lymphocytes).

In one embodiment of the invention, the ascorbic acid may induce the activity of the immune response by maturation of dendritic cells in an animal having a tumor.

In addition, the present invention provides a vaccine composition comprising the adjuvant composition and the target antigen as an active ingredient.

In one embodiment of the present invention, the antigen may be a protein, a recombinant protein, a glycoprotein, a peptide, a polysaccharide, a peptide or a polynucleotide, but is not limited thereto. For example, in the case of a mouse, OVA (ovalbumin) specific for a mouse can be included as an antigen, but is not limited thereto.

In one embodiment of the present invention, the antigen may be a cell, a bacteria or a virus.

In one embodiment of the present invention, the antigen may be a tumor-specific antigen. For example, the antigen is a tumor-specific antigen, which promotes an immune response to an antigen in the body, thereby inhibiting the growth of cancer cells when cancer cells expressing the antigen are developed, thereby preventing or treating cancer have.

In one embodiment of the present invention, the vaccine composition may be one that induces the production of an antibody specific for the target antigen.

In one embodiment of the present invention, the vaccine composition may induce the activity of CTL (cytotoxic T lymphocytes) specific to the target antigen.

The present invention also provides a composition for preventing or treating cancer comprising the vaccine composition as an active ingredient.

The present invention also provides a pharmaceutical composition for inhibiting recurrence or metastasis of cancer comprising the vaccine composition as an active ingredient.

The present invention provides a pharmaceutical composition comprising a vaccine and a pharmaceutically acceptable vehicle, excipient or carrier. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, the vehicle may contain wetting or emulsifying agents, pH buffering agents, or other excipients such as adjuvants. Pharmaceutically acceptable carriers may contain physiologically acceptable compounds which act on, for example, stabilize, increase or decrease the absorption or removal rate of the pharmaceutical compositions of the present invention. Physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, detergents, liposome carriers or other stabilizers and / Or buffers. Excipients can be nonionic surfactants, polyvinylpyrrolidone, human serum albumin, aluminum hydroxide, anesthetic agents, and a variety of unmodified derivatized cyclodextrins. More preferably, the nonionic surfactant may comprise polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. The polyvinylpyrrolidone may be Plasdone C15, preferably a pharmaceutical grade polyvinylpyrrolidone.

The anesthetic agent is preferably benzyl alcohol. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives. The pharmaceutical compositions of the present invention may include adjuvants such as pharmacological agents, cytokines, or other biological response modifiers. Pharmaceutical compositions comprising such excipients or carriers are formulated by any of the well-known conventional methods.

The vaccine according to the present invention may be formulated for the following routes of administration: intramuscular, intradermal, oral, transdermal, nasal, oral, rectal, vaginal, by inhalation, or by subcutaneous administration. Other modes of administration may be applied as long as satisfactory immunogenicity can be induced.

The pharmaceutical compositions of the present invention may be prepared injectable, either as a liquid solution or suspension, or in solid form suitable for being dissolved or suspended in a liquid vehicle prior to injection. The pharmaceutical compositions may also be prepared as an active ingredient encapsulated in a liposomal vehicle or other particulate carrier for use in emulsified solid forms or sustained delivery. For example, the pharmaceutical composition may be in the form of an oil-in-water emulsion, a water-in-oil emulsion, an oil-in-water emulsion, a site-specific emulsion, an organ- , Nanoparticles and various natural or synthetic polymers such as non-resorbable impermeable polymers such as ethylene vinyl acetate copolymers and Hytrel copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or Polyesters, such as those used to make resorbable sutures that allow for sustained release of the vaccine.

In one embodiment of the invention, the cancer is selected from the group consisting of breast cancer, lung cancer, esophageal cancer, rectal cancer, biliary cancer, liver cancer, bowel cancer, stomach cancer, colon cancer, pancreatic cancer, kidney / kidney cancer, (For example, basal cell carcinoma, squamous cell carcinoma, or melanoma), for example, cancer, endometrial cancer, endometrial cancer, pancreatic cancer, testicular cancer, bladder cancer, head and neck cancer, oral cancer, neuroendocrine cancer, adrenocortical cancer, But is not limited thereto.

In the present invention, an effective amount of a vaccine is an amount capable of inducing an immunological effect, such as stimulating IgG production, against a target antigen. The effective amount or dose of the vaccine will depend on the amount of target antigen, the type of adjuvant, the mode of administration, and the age, size and condition of the subject being treated.

The vaccine may be administered at a stat dose with or without one or more additional doses at specific time intervals to achieve long term immunoprotective effects from months to years. The frequency of administration may depend on any of a variety of factors such as the severity of the symptoms, the severity of the desired immune protection, whether the pharmaceutical composition has been used for prophylactic or therapeutic purposes, and the like. For example, in one embodiment, the pharmaceutical composition of the present invention is administered once a month, twice a month, three times a month, every other week, qw every week, biw every week, three times a week, (Qid), twice daily (qd), twice daily (qid), or three times a day (tid) daily, twice a week. The vaccine may also be administered concurrently or sequentially with other conventional therapies, such as antibodies that target chemotherapy, targeted therapies, or tumor-associated carbohydrate antigens for cancer therapy.

Ascopyran according to the present invention can be used as an adjuvant by inducing maturation of dendritic cells in the spleen and lymph nodes in an animal having a tumor and by increasing the immune response and CTL activity of Th1 cell mediated by mature dendritic cells, The adjuvant and the tumor-specific antigen may be administered together as a vaccine capable of inducing immunization to induce the immune response, thereby being useful for prevention or treatment of cancer as well as inhibiting recurrence or metastasis of cancer.

Brief Description of the Drawings Figure 1 shows the effect of ascopyran on in vivo &Lt; / RTI &gt; (A) is a lineage in the spleen and tumor drLN ^- is a result of analyzing the percentage of CD11c ⁺ DC in the flow cytometry, (B) the spleen (left panel) and tumor drLN in living cells (right panel), lineage ^- CD11c ⁺ (C) is the measurement of the expression level of CCR7 in lineage ^- CD11c ⁺ DC in spleen and tumor drLN, (D) shows the expression level of CCL19 and CCL21 in splenocytes (E) is the result of real-time PCR analysis, (E) is the result of labeling the DC separated from the mouse with CellVeu Maroon and analyzing the movement of labeled DC by flow cytometry, and (F) co in the CD11c ⁺ DC - ^- lineage will showing the flow cytometric analysis of stimulatory molecules, MHC class I and II, (G) is the result of measuring the amount of IL-6, IL-12p70 and TNF-α in the serum, (H) is the result of analysis of the production of L-6, IL-12 and TNF-α in cells of spleen DC (* p <0.05 , ** p < 0.01 ).
Figure 2 shows the results of promoting Th1 and Tc1 immune responses by ascorbic acid in mice with tumors. (A) expresses IFN- [gamma] ⁺ in CD4 ⁺ and CD8 ⁺ T cells within spleen and tumor drLN, ^{IL-17 +, TNF-α} +, and IL-4 will showing the percentage of ^+, (B) the spleen (upper panel) and tumor drLN IFN-γ in the living cell at the (lower panel) ⁺ and TNF-α ⁺ will showing the absolute average number of the cells, (C) shows the amount of IFN-γ and TNF-α in the serum, (D) is in the spleen cells T-bet, IFN-γ, IL-4 and The expression level of IL-17A was analyzed by real-time PCR (** p <0.01 ).
Figure 3 shows the results for antigen-presenting CD4 and CD8 T cell proliferation by ascorbic acid in mice with tumors. (A) is the result of analysis of the percentage of lineage ^- CD11c ⁺ CD8α ⁺ and lineage ^- CD11c ⁺ CD8α ^- cDC in the spleen by flow cytometry (upper panel) and the expression level of MHC class I and II (lower panel) , (B) is a CD11c ⁺ CD8α ⁺ and CD11c ⁺ CD8α in the living cells in the spleen ^- will shown the absolute number average of the cDC, (C) the CD11c ⁺ CD8α ⁺ and CD11c ⁺ CD8α ^- presentation of the OVA peptide from cDC (presentation (D) shows the results obtained by labeling CD8 T cells or CD4 T cells isolated from OT-I or OT-II mice with CFSE and comparing CD45 T cells with B16 tumors (E) shows that OT-I cells (left panel) and OT-II cells (left panel) in the tumors were differentiated into CFSE dilution (Right panel), and (F) is the average of the absolute numbers Tumor-infiltrating from the OT-I cells and OT-II shows the results of analysis of IFN-γ ⁺ TNF-α ⁺ and percentage of cells (** p <0.01, * p <0.05).
Figure 4 shows the inhibitory effect of B16-OVA tumor growth by treatment with the combination of ascorbyl-OVA. (A) shows the B16-OVA tumor growth curve implanted on the right side of the mouse, (B) shows the size of the tumor mass measured on the 28th day after B16-OVA tumor cell transplantation, (D) shows the tumor growth cuff grafted to the left side on the left side after 16 days of inoculation with B16-OVA cells on the right side of the mouse, and (E) shows survival rate (F) represents the percentage of mice with tumors on the left side of the tumor (** P <0.01 ).
Figure 5 shows the results of the adjuvant effect of ascopyran on OVA-induced B and T cell immune responses. (A) is the result of ELISA (OVA-specific IgG1 (left panel) and IgG2 (right panel) concentration at the 35th day after immunization ( * P <0.05 in the OVA group , ** P < 0.01 ) , # P < 0.05, # P < 0.01 in PBS group and (B) at day 35 after immunization, spleen cells were re-stimulated with OVA (50 μg / ml) (C) is a result showing the IFN-γ concentration in the cultured supernatant of the spleen cells, (D) shows the amount of CD44 expression in the CD4 or CD8 T cells, (Left panel), which is the mean percentage of CD44 ⁺ cells (right panel) (** p <0.01 )
Figure 6 shows the inhibitory effect of Ascopyran-OVA on cancer formation by B16-OVA melanoma cells by immunization. (A) depicts the mawooseuyi percent with a tumor, (B) will showing a state of mice with tumors in 28 days, (C) will showing tumor growth curve (* P <0.01), ( D) Was the result of in vivo CTL activity measured by flow cytometry at day 35 after immunization and dotplot was the result of the percent of SIINFEK-injected CFSE ⁺ cells and non-peptide-injected CMTMR ⁺ cells Panel), and the mean percentage value of antigen-specific cell lysis (bottom panel) (** p &lt; 0.01 ).
Figure 7 shows the inhibitory effect of B16-OVA melanoma metastasis to liver by ascorbyl-OVA immunization. (A) represents the survival rate of the mouse, (B) represents the size of the tumor mass in the spleen and the metastasis of B16-OVA cells to the liver on the 10th day after tumor injection, and (C) The mean absolute value of the -OVA transition is (** p <0.01 ).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1. Materials and Methods

1.1. Mouse and cell line

C57BL / 6 mice (6 weeks old), OT-I and OT-II TCR transgenic mice and C57BL / 6-Ly5.1 (CD45.1) congenic mice were obtained from Shanghai Public Health Clinical Center (Shanghai, CN) and maintained in a pathogen-free environment. Mice were freely accessible to standard rodent food and water and maintained at the conditioned temperature (20-22 C), humidity (50-60%) and light (12 h name: 12 h cancer) Experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee in the Shanghai Public Health Clinical Center. The protocol was approved by the Animal Experimental Ethics Committee of the Shanghai Public Health Clinical Center (Mouse Protocol Number: SYXK-2010-0098). The mice were euthanized by inhalation of CO ₂ , and the logo worked to minimize pain. (10% FBS, 2 mM glutamine, 1 M HEPES, 100 μg / ml streptomycin, 100 μg / ml) were cultured in RPMI 1640 (Sigma Aldrich) to express ovalbumin U / ml penicillin, and 2 mM 2-mercaptoethanol). All cell lines were cultured at 37 ° C in a 5% CO ₂ incubator.

1.2. chemical substance

Ascophyllan was prepared from pulverized Ascophyllum nodosum as previously described (Nakayasu S, Soegima R, Yamaguchi K and Oda T. Bioscience, Biotechnology, and Biochemistry 2009; 73 (4): 961-964; and Jiang Z, Okimura T, Yamaguchi K and Oda T. Biology and Chemistry / Official Journal of the Nitric Oxide Society 2011; 25 (4): 407-415). The ascorbyl solution was passed through an endotoxin-removing column (Detoxi-gel: Thermo Fisher Scientific) and sequentially passed through an endotoxin-removing filter (Zetapor Dispo: Wako). The amount of endotoxin in the purified ascorbic acid was evaluated using the LAL (Limulus amebocyte lysate) assay kit (Lonza). ^{H2-K b restricted peptide OVA257-264 (} SIINFEKL; SEQ ID NO: 1) was purchased from Chinapeptides (China).

1.3. Antibody

Isotype control Abs (antibodies) (IgG1, IgG2a or IgG2b), CD11c (HL3), CD4 (GK1.5), CD8a (YTS169.4), CD40 (3/23), CD80 -1), CCR7 (4B12), anti-IL-4 (11B11), anti-IL-6 (MP5-20F3) and anti-IL-12 / 23p40 (C17.8) were purchased from BioLegend; anti-IL-17 (TCC11-18H10.1) and anti-IL-17 (anti-IL-17) Anti-TNF-α (MP6-XT22) was purchased from eBioscience.

1.4. Flow cytometry

Cells were washed in PBS (phosphate buffered saline) containing 0.5% BSA, pretreated with unlabeled isotype control Abs for 15 min, labeled with fluorescence-conjugated Abs in ice cube for 30 min and washed with PBS . Cells were analyzed with FACS Aria II (Becton Dickinson) and FlowJo 8.6 software (Tree Star). Cell debris was excluded from analysis by forward and side-scatter gating. Dead cells were also excluded by 7AAD (7 aminoadinomycin D; BioLegend) staining and gating for the 7AAD-negative population. As a control for non-specific staining, isotype-matched irrelevant mAbs were used.

1.5. DC ( dendritic  cell analysis

DCs (dendritic cells) of spleen and tumor draining lymph node (DRLN) were analyzed as reported previously (Jin JO, Zhang W, Du JY, Wong KW, Oda T and Yu Q. PloS One, 2014; 9 (6): e99396; and Zhang W, Du JY, Jiang Z, Okimura T, Oda T, Yu Q and Jin JO., Marine Drugs 2014, 12 (7): 4148-4164). Briefly, the tissue was cut into small pieces and digested with 2% FBS (fetal bovine serum) containing collagenase for 20 minutes at room temperature. The digested cells were centrifuged and the pellet was redissolved in 5 ml of histopaque (Sigma-Aldrich). Subsequently, the histopaque added by centrifugation at 1700 g for 10 minutes consisted of the lower layer and EDTA-FBS was placed in the upper layer. A light density fraction (<1.077 g / cm ³ ) was collected and treated with the following FITC-conjugated mAbs (monoclonal antibodies) for 30 min: anti-CD3 (17A2), anti-Thy1.1 -7), anti-B220 (RA3-6B2), anti-Gr1 (RB68C5), anti-CD49b (DX5) and anti-TER-119 (TER-119). lineage ^- CD11c ⁺ cells were defined as cDCs and classified as CD8α ⁺ and CD8α ^- cDCs. Analysis was performed with FACS Aria II (Becton Dickinson).

1.6. DC ( dendritic  cell movement Assay

CD11c ⁺ spleen DCs were isolated by biotin-conjugated anti-CD11c Abs (Miltenyibiotec) in C57BL / 6 mice and the cells were labeled with the CellVue Maroon cell labeling kit (eBioscience). The labeled cells were then transferred to tumor-bearing mice, and the mice were treated with or without ascorbyl-treatment after 1 hour of DC delivery. Cells transferred to the spleen and tumor drLN (tumor draining lymph node) were analyzed by flow cytometry from lineage ^- CD11c ⁺ cells.

1.7. Ex vivo  T cell stimulation and intracellular cytokine staining

Single cell suspensions prepared from spleen and tumor drLN were cultured in the presence of PMA (phorbol 12-myristate 13-acetate, 50 ng) as described in detail previously (Jin JO, Han X and Yu Q. Journal of autoimmunity. 2013; 40: / ml; Calbiochem) and ionomycin (ionomycin, 1 μM; Calbiochem) was stimulated by both in vitro for four hours, during the final two hours the parent nensin solution (monensin solution; was added Biolegend). Positive controls were stimulated with soluble anti-CD3 (1 μg / ml) and anti-CD28 (1 μg / ml) for 3 days in splenocytes (2 × 10 ⁶ / ml) from C57BL / 6 mice . IL-4 (Peprotech) or IL-6 (Peprotech) was added to the culture medium. Thereafter, the production of IL-4 and IL-17 in the cells was measured, respectively. For cytokine staining in cells, cells were first stained with surface molecules, immobilized and permeabilized with Cytofix / Cytoperm buffer (eBioscience), and sequentially incubated for 30 minutes with an anti-cytokine antibody in Perm / Wash buffer (eBioscience) Lt; / RTI &gt; Control staining with isotype control IgGs was performed in all experiments.

1.8. ELISA

Serum levels of IL-6, IL-12p70, IFN-γ and TNF-α were measured three times with standard ELISA kits (Biolegend).

1.9. Real-time PCR

Total RNA was reverse transcribed using Oligo (dT) and M-MLV reverse transcriptase (Promega). The cDNA was amplified by real-time PCR amplification kit (Qiagen) and annealed at 60 ° C for 40 cycles. LightCycler 480 Real-Time PCR System (Roche) was used. Primer sequences are shown in Table 1 below.

gene Primer direction SEQ ID NO: mouse β-actin forward 5'-TGGATGACGATATCGCTGCG-3 '(SEQ ID NO: 2) reverse 5'-AGGGTCAGGATACCTCTCTT-3 '(SEQ ID NO: 3) CCL19 forward 5'-CTGCCTCAGATTATCTGCCAT-3 '(SEQ ID NO: 4) reverse 5'-AGGTAGCGGAAGGCTTTCAC-3 '(SEQ ID NO: 5) CCL21a forward 5'-ATCCCGGCAATCCTGTTCTC-3 '(SEQ ID NO: 6) reverse 5'-GGTTCTGCACCCAGCCTTC-3 '(SEQ ID NO: 7) T-bet forward 5'-CAACAACCCCTTTGCCAAAG-3 '(SEQ ID NO: 8) reverse 5'-TCCCCCAAGCATTGACAGT-3 '(SEQ ID NO: 9) IFN-y forward 5'-GGATGCATTCATGAGTATTGC-3 '(SEQ ID NO: 10) reverse 5'-CTTTTCCGCTTCCTGAGG-3 '(SEQ ID NO: 11) IL-4 forward 5'-ACAGGAGAAGGGACGCCAT-3 '(SEQ ID NO: 12) reverse 5'-GAAGCCCTACAGACGAGCTCA-3 '(SEQ ID NO: 13) IL-17A forward 5'-GCGCAAAAGTGAGCTCCAGA-3 '(SEQ ID NO: 14) reverse 5'-ACAGAGGGATATCTATCAGGG-3 '(SEQ ID NO: 15)

1.10. Mouse immunization

C57BL / 6 mice were treated with PBS alone; 2.5 mg / kg of OVA (in PBS); 50 mg / kg ascopyran (in PBS); Or OVA-ascorbyl (in PBS) at 0, 15, and 30 days ( iv ). On day 35, mice were sacrificed to collect serum, and splenocytes were isolated for further analysis. Some of the mice were formed with B16-OVA melanoma.

1.11. OVA-specific antibody analysis

The 96-well plate was coated with OVA (10 μg / ml) as described previously (Jin JO, Zhang W, Du JY, Wong KW, Oda T and Yu Q. PloS one. ) And blocked with 1% BSA (bovine serum albumin). Serum samples were diluted and added to each well, and biotin-conjugated anti-mouse IgG1 and IgG2a (Biolegend) and streptavidin-conjugated HRP (Horseradish peroxidase) were added. Reactions were developed with TMB substrate (Sigma) and A ₆₅₀ was measured using a plate reader.

1.12. OT -I and OT -II T cell proliferation

CD8 T cells from CD4 T cells or OT-I mice from OT-II mice were each isolated from the spleen using CD4 T cell or CD8 T cell isolation kit (Miltenyi Biotec). Cells were suspended in 0.1% BSA (in PBS) containing 10 μM CFSE (Invitrogen) and incubated for 10 min. CFSE-labeled cells (1 × 10 ⁶ ) were delivered intravenously ( iv ) to CD45.1 conscious mice, and after 24 h, PBS alone; 2.5 mg / kg of OVA (in PBS); Or OVA-ascorbic acid (in PBS). Splenocytes were collected at 72 hours after immunization and OT-I or OT-II T cell proliferation was determined by analyzing the CFSE fluorescence density through a flow cytometer.

1.13. In vivo  Cytotoxicity analysis

Mice were injected intravenously with a mixture of splenocytes labeled with CFSE (2, 20 or 200 nM), and 1, 10, or 100 nM SIINFEKL peptide was injected. In addition, the peptide was not injected into mice injected with spleen cells labeled with 10 mM CellTracker ^™ Orange CMTMR (Life technologies). A total of 10 x ¹⁰⁶ cells per mouse were injected, which consisted of a mixture containing each target cell group. Splenocytes were collected 24 hours after injection of the target cells. The presence of surviving target cells was determined to be exclusion by 7-AAD (7-aminoactinomycin D). Percentage of dead cells was calculated using the previously described formula (Hermans IF, Silk JD, Yang J, Palmowski MJ, Gilead U, McCarthy C, Salio M, Ronchese F and Cerundolo V. Journal of immunological methods. (1): 25-40).

1.14. Spleen ( intrasplenic ) Injection

C57BL / 6 mice were anesthetized by injecting a ketamine mixture (10 μL ketamine HCl, 7.6 μL xylazine, 2.4 μL acepromazine maleate, and 10 μL H ₂ O) into the peritoneal cavity. B16-OVA melanoma cells ^{(0.5 x 10 6/50 μL} ) is open laparotomy for the experiment was injected into the mouse spleen for the (open laparotomy).

1.15. Statistical analysis

The results were expressed as mean ± SEM (standard error of the mean). Statistical significance between the experimental groups was calculated using the Bonferroni post-test or unpaired Student's t-test using analysis of variance. p-value <0.05 was considered significant.

Example  2. In the tumor environment Ascopilan  Induced DC activity

In our previous studies, it has been shown that ascopyran induces DC maturation of the spleen (Zhang W, Du JY, Jiang Z, Okimura T, Oda T, Yu Q and Jin JO, Marine drugs 2014, : 4148-4164). In the present invention, it was confirmed whether ascorbicin induces DC maturation in tumor-bearing mice. C57BL / 6 mice were injected subcutaneously ( sc ) with 1 x ¹⁰⁶ B16 melanoma cells. When tumors were well formed, mice with tumors were injected intravenously ( iv ) with 50 mg / kg of ascorbic acid and analyzed the activity of DCs in the spleen and tumor drLN after 24 hours. Treatment of ascorbic acid induced a significant increase in the ratio and number of DCs in the spleen and tumor drLN (FIGS. 1A and 1B). (CC motif ligand 19) and CCL21 in the spleen are present in spleen DC and tumor drLN, and in the spleen, The expression level of the ligand CCR7 (CC chemokine receptor 7) was significantly increased by ascorbic acid (FIGS. 1C and 1D). To determine if Ascopilan promotes DC migration, we transferred CellVue Maroon-labeled DCs to mice with tumors and treated the Ascopilans two hours after delivery. Furthermore, the expression levels of co-promoting molecules and MHC class I and II in the DCs of spleen and tumor drLN were significantly up-regulated by ascorbic acid (Fig. 1F).

It is well known that mature DCs secrete pro-inflammatory cytokines including IL-6, IL-12 and TNF-a (Banchereau J and Palucka AK. Nature Reviews Immunology. 2005; 5 (4): 296- 306). Thus, the present inventors have tested whether ascopyran can promote the production of pro-inflammatory cytokines by DCs in mice bearing tumors. The amount of serum cytokine was analyzed 24 hours after the administration of ascorbic acid to the tumor-bearing mice. As in FIG. 1G, the amounts of IL-6, IL-12p70 and TNF-a were significantly increased compared to mice with PBS treated tumors. We have analyzed the production of intracellular cytokines in splenic DCs to determine if the cytokines are produced by DCs stimulated by ascorbyl. As in Figure 1, the treatment of ascorbic acid induced a significant increase in the percentage of DC producing IL-6, IL-12 and TNF- alpha in the spleen of mice bearing tumors.

Thus, it has been shown that ascopyllan can induce the migration of DCs into spleen and tumor drLN in mice with tumors and can promote the activity of DCs.

Example  3. With tumor  In tumor-bearing mice Ascopilan  Th1 and Tc1  Promotion of response

Since Ascopyran induces DC activity in mice with tumors, we tested whether DC activity of ascorbyl-inducing in tumor-bearing mice could promote T cell immune response. When formed in B16 black paper mice, the mice were intravenously ( iv ) injected twice at 3-day intervals with 50 mg / kg ascopyran and analyzed 3 days after the second injection. Treatment of ascorbicin induced a significant increase in the ratio of CD4 and CD8 T cells producing IFN-y and TNF-a in spleen and tumor drLN, whereas CD4 and CD8 producing IL-4 or IL-17 T cells were not increased by treatment with ascorbic acid (Fig. 2A). Consistent with flow cytometry data, the number of CD4 and CD8 T cells producing IFN-y and TNF-a in spleen and tumor drLN was significantly increased by treatment with ascorbic acid (Fig. 2B). In addition, serum levels of IFN-y and TNF-a were significantly elevated by treatment with ascorbic acid in mice with tumors (Fig. 2C). Moreover, the amount of mRNA expression of T-bet (an important transcription factor for Th1 and Tc1 cells) and IFN-y in splenocytes was significantly increased by treatment with ascorbic acid, whereas the expression level of IL-4 and IL-17A mRNA But not by ascorbic acid (Fig. 2D).

Thus, it has been shown that treatment with ascorbic acid can promote Th1 and Tc1 immune responses in mice with tumors.

Example  4. With tumor  In tumor-bearing mice Ascopilan  &Lt; / RTI &gt; antigen presentation and antigen-specific enhancement of T cell proliferation

Other sets of DCs have been shown to exhibit other specialized functions in antigen presentation (Schnorrer P et al., Proceedings of the National Academy of Sciences, 2006; 103 (28): 10729-10734) . To determine whether ascopyran can promote antigen presentation or cross-presentation in mice with tumors, we inject PBS, OVA (ovalbumin) or Ascopyran-OVA into mice with tumors Respectively. After 24 hours, the expression levels of MHC class I and II were measured in spleen CD8 alpha ⁺ and CD8 alpha ^- cDC.

As a result, the combination of Ascopilan and OVA promoted significant upregulation of MHC class I and II expression in both CD8α ⁺ and CD8α ^- cDC compared to mice treated with OVA alone (FIG. 3A). In addition, the number of CD8? ⁺ And CD8 ^? -CDCs in the spleen was significantly increased by the combination of ascorbyl ^- OVA (FIG. 3B). CD8a ⁺ and CD8a ^- cDCs treated with the Ascopyran-OVA combination showed a higher percentage of OVA peptide presentation than with OVA or Ascopilane alone (Figure 3C).

Next, in order to determine antigen-specific T cell proliferation, we delivered CFSE-labeled OT-I and OT-II cells to CD45.1 conical mice with tumors, and after 24 hours, , OVA, ascorbic acid, or ascorbyl-OVA for 3 days. The combination of Ascopyran-OVA, but not OVA and Ascopyllan alone, induced a significant increase in the proliferation of OT-I and OT-II T cells in the spleen (Figure 3D). In addition, the number of OT-I and OT-II cells in which tumors were intro- duced was strongly increased in mice treated with a combination of ascorbyl-OVA (Fig. 3E). Furthermore, the intracellular production of IFN-? And TNF-? In OT-I and OT-II cells in which tumors were intruded in mice treated with a combination of ascopyran-OVA were compared with those treated with ascopyran or OVA alone (Fig. 3F).

Thus, it has been shown that ascopyran promotes antigen-specific T cell responses in tumor-bearing mice and functions as an immunogenic adjuvant.

Example  5. Ascopilan -OVA Immunization to Inhibit the Growth of B16-OVA Tumor Cells

Because ascopyran promotes DC and antigen-specific T cell activity in mice with tumors, the inventors have experimented that ascopyran can be used as an effective adjuvant in tumor immunotherapeutic agents. First, we evaluated the effect of ascorbyl as an adjuvant for therapeutic vaccination in mice with tumors. C57BL / 6 mice were inoculated subcutaneously ( sc ) with 1 x 10 ⁶ B16-OVA melanoma cells on the right side of the mice. After 7 days, when the tumor was well formed, the mice were intravenously injected with PBS, OVA (ovalbumin), ascorbicin or ascorbyl-OVA, and treated again with the same reagent after 7 days. On the 16th day after inoculation of early tumor cells, the same number of B16-OVA cells were inoculated again into the left side of the mouse.

As a result, treatment with the combination of ascorbyl-OVA markedly inhibited the growth of the B16-OVA tumor on the right side of the mouse (Fig. 4A). As in FIG. 4B, mice treated with the Ascopilan-OVA combination showed a significantly smaller tumor mass at 28 days when compared to mice treated with OVA or ascorbicin alone. Furthermore, mice treated with a combination of ascorbyl-OVA survived longer than mice treated with PBS, OVA or ascorbic acid alone (FIG. 4C). In addition, in the mice treated with OVA or ascorbicin alone, a second tumor-derived left mass was identified on the 28th day after the initial tumor formation on the right side, whereas mice treated with the Ascopyran-OVA combination No second tumors were completely formed (Fig. 4D, 4E and 4F). This means that the combination of ascorbyl-OVA induces the formation of a long-term memory immune response to the OVA antigen.

Thus, it has been found that ascopyran can function as a therapeutic vaccine adjuvant.

Example  6. in vivo To improve the OVA-specific immune response in As an adjuvant  Functional Ascopilan  effect

From the discovery that ascopyran-OVA treatment inhibits the formation of a second tumor in the mouse, we examined whether OVA-immunization with ascorbicin could promote OVA-specific antibodies and T cell immune responses. C57BL / 6 mice were intravenously ( iv ) injected with OVA alone or 50 mg / kg ascopyran at 0, 15 and 30 days. On day 35, serum and spleen cells were collected for analysis. OVA-immunization with ascorbic acid induced a significant increase in the amount of OVA-specific IgG1 (IgG1) and IgG2a in serum as compared to PBS treatment, whereas immunization with OVA alone had the effect described above (Fig. 5A).

At day 35, spleen cells were stimulated again in vitro with OVA for 4 days and T cell immune response against OVA was measured. Splenocytes derived from mice immunized with the combination of Ascopilan-OVA showed significantly increased cell proliferation and IFN-y production (Fig. 5B and 5C) as compared to spleen cells derived from mice immunized with OVA alone. Moreover, the expression level of CD44, an important surface marker of effector / memory T cells, is significantly higher than that of mice immunized with a combination of ascopyran-OVA versus spleen CD4 and CD8 T cells derived from mice treated with PBS or OVA alone And was significantly higher in spleen CD4 and CD8 T cells (Fig. 5D).

Thus, it was confirmed that ascopyran functions as an adjuvant capable of enhancing antigen-specific B cell production and T cell immune response.

Example  7. Ascopilan - Inhibitory effect of B16-OVA tumor on OVA immunization

From the discovery that ascopyran can function as an adjuvant for an OVA-specific immune response, the present inventors have made it clear that the immune response can protect mice transplanted with B16-OVA tumor cells. C57BL / 6 mice were immunized intravenously ( iv ) with a combination of PBS, OVA, ascorbyllan or ascorbyl-OVA at 0, 15, 30 days. On day 35, B16-OVA melanoma cells were inoculated subcutaneously ( sc ) in mice. As a result, it was confirmed that the mice immunized with the Ascopyran-OVA combination were almost completely protected from B16-OVA tumor formation (Figs. 6A and 6B). Furthermore, mice immunized with the Ascopyran-OVA combination showed significantly reduced tumor growth compared to mice immunized with PBS, OVA or ascorbicin alone (FIG. 6C).

Next, we investigated the functional activity of CTL (cytotoxic T lymphocytes) in an in vivo cytotoxicity assay. After the initial immunization, the mice immunized in the second 35 days and C57BL / 6 donor (donor) measuring the specific cytolytic intravenous (iv) was injected, by a flow cytometer to SIINFEKL- pulses and CFSE- labeled splenocytes from mice Respectively. Specific target cell lysis was 75% in mice immunized with the Ascopyran-OVA combination, indicating T cell memory induction (Figure 6D). In contrast, mice immunized by OVA or ascorbicin alone did not show significant killing of OVA-pulsed splenocytes (Fig. 6D).

Thus, ascopyran is expressed in vivo To promote the formation of antigen-specific immune responses in tumors in the long term and to protect mice against tumor formation.

Example  8. Ascopilan Inhibitory effect of B16-OVA melanoma metastasis on OVA immunization

Next, the present inventors confirmed whether treatment with Ascopyran-OVA combination can inhibit tumor metastasis in mice. Mice PBS, was 50 μg of the OVA, 50 mg / kg of ascorbyl pilran or ascorbyl pilran -OVA injected intravenously (iv), three days later in the spleen to 0.5 x 10 ⁶ B16-OVA melanoma cells (is.) In Respectively. Three days after tumor cell injection, mice were treated with the same amount of ascorbyl-OVA.

As a result, mice treated with PBS, OVA or ascorbic acid alone began to die from day 14 after inoculation with B16-OVA cells, and all mice died within 18 days. In contrast, mice treated with the Ascopilan-OVA combination began to die from day 26 after B16-OVA inoculation and all mice died within 28 days (Figure 7A). Moreover, the size of tumor mass on the spleen 10 days after injection of B16-OVA cells was much smaller in mice treated with the Ascopyran-OVA combination compared to mice treated with OVA or ascorbic acid alone 7B). In addition, mice treated with the Ascopilan-OVA combination showed very low degrees of tumor cell invasion and metastasis (observed in all mice) to the liver as compared to the other experimental groups (Figures 7B and 7C).

Thus, it has been shown that the Ascopyran-OVA combination can protect against invasion and metastasis of B16-OVA melanoma cells into the liver.

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Claims (14)

delete delete delete delete delete delete 1. A cancer vaccine composition comprising an ascorbyl adduct composition and an OVA257-264 peptide represented by SEQ ID NO: 1 as an active ingredient. delete delete 8. The method of claim 7,
Wherein said OVA257-264 peptide is an antigen specific for a tumor. 8. The method of claim 7,
Wherein said vaccine composition induces the production of an antibody specific for said OVA257-264 peptide. 8. The method of claim 7,
Wherein said vaccine composition induces the activity of CTL (Cytotoxic T lymphocytes) specific to said OVA257-264 peptide. A pharmaceutical composition for preventing or treating cancer comprising the vaccine composition of claim 7 as an active ingredient. A pharmaceutical composition for inhibiting recurrence or metastasis of cancer comprising the vaccine composition of claim 7 as an active ingredient.

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