KR20120113823A - Pharmaceutical composition for enhancing immune function comprising baccatin iii as active ingredient - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing baccatin III is provided to promote antigen processing in antigen presenting cells(APC) and to improve antigen-specific CTL reaction. CONSTITUTION: A pharmaceutical composition for enhancing immunity contains pharmaceutically effective amount of baccatin III and pharmaceutically acceptable carriers. Baccatin III enhance antigen presenting level through MHC(major histocompatibility complex) in antigen presenting cells(APC). Baccatin III improves antigen-specific CTL(cytotoxic T lymphocyte) reaction inducing ability.

Description

Pharmaceutical composition for Enhancing Immune Function Comprising Baccatin III As Active Ingredient}

The present invention relates to a pharmaceutical composition for enhancing immunity comprising baccatin III as an active ingredient.

In order for activation of T cells and subsequent effective functions of T cells to occur, at least two signals transmitted by antigen presenting cells (APCs) are required. One of the signals is an antigen specific signal (signal-1) transmitted through the MHC / peptide structure, and the other signal is a costimulatory signal (co a stimulatory signal (signal-2) [1]. The concept of co-stimulation was introduced in 1980, and it has been recognized that blocking or enhancing co-stimulation signals is a very attractive strategy for inducing immune tolerance or activating various immune responses [2-4]. It is well known that stimulation of T cells by antigen in the absence of co-stimulation leads to functionally non-responsive or clonal anergy [5, 6]. Antigen-specific signals provided by peptides complexed with MHC molecules received little attention as targets for therapeutic immunomodulation despite the fact that they account for almost half of the T cell activation process. Since T cells recognize only antigens expressed on MHC molecules [7], strategies for activating MHC-restricted antigen processing pathways are directed at therapeutic immunoactivation, in particular antigen-specific signals delivered by MHC / peptide structures. New means may be provided in situations that fall short of the limitations of T cell priming. Various evidences demonstrate that MHC-restricted antigen presentation can be upregulated or downregulated by drugs. Chloroquinone or ammonium chloride, which acts to reduce acidity in the endocytic compartment, has been reported to increase class I MHC-restricted external antigen presentation [8]. Modification of the antigen by hypochloric acid (HOCl), a major oxidant produced by neutrophils, has been shown to increase the presentation of class I and class II MHC-restricted antigens [9]. Meanwhile, it has already been reported that cyclosporin A and tacrolimus, the major drugs that have been used to inhibit transplant rejection, severely inhibit class I and class II MHC-limiting presentation of antigenic peptides in both in vitro and in vivo. This suggests that the immunosuppressive activity of cyclosporin A and tacrolimus is at least in part due to inhibition of the antigen processing pathway [10-12].

Simvastatin, an inhibitor of the rate-limiting enzyme HMG-CoA reductase of the cholesterol biosynthetic pathway, has been reported to inhibit class I MHC-restricted presentation of exogenous antigens [13]. Inhibition of MHC-restricted antigen presentation has also been reported for nonsteroidal anti-inflammatory drugs such as aspirin and ibuprofen [14]. These reports also suggest that regulating antigen-specific signals may allow older drug molecules to become new targets for the therapeutic regulation of T cell responses.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The inventors have tried to search for therapeutic immunomodulators through T cells. As a result, baccatin III, a semisynthetic precursor of the anticancer drug paclitaxel, promotes the processing of internal and external antigens in antigen-presenting cells, significantly increases the presentation of antigen by MHC, and antigen-specific. By improving the induction of the Cytotoxic T Lymphocyte (CTL) response, the present invention was completed by experimentally confirming that it has potent immunopotentiating activity.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for immune enhancement comprising bacatin III as an active ingredient.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of baccatin III; And (b) a pharmaceutically acceptable carrier.

According to the present invention, bacterin III, a semisynthetic precursor of paclitaxel, promotes the processing of internal and external antigens in antigen-presenting cells, significantly increases the presentation of antigens by MHC, and antigen-specific Cytotoxic T Lymphocyte (CTL). It is based on the discovery that it has strong immunopotentiating activity by enhancing the induction of the response.

The active ingredient Bacatin III of the present invention is a compound known as a semisynthetic precursor of paclitaxel, an anticancer agent, and is a compound represented by the following Chemical Formula 1.

According to a preferred embodiment of the present invention, the bacterin III greatly increases the level of antigen presentation through the major histocompatibility complex (MHC) in the antigen presenting cell.

As used herein, the term “antigen presenting cell” refers to the processing of an antigen into the form of a peptide fragment, which binds to the antigenic peptide binding site of the MHC molecule to bind T cells with the MHC molecule at the cell surface. Means the cells to present. Preferably, the antigen presenting cell is a dendritic cell (Dendritic Cell, DC).

As used herein, the term “processing” of an antigen is in the form of a small size fragment suitable for binding an exogeneous antigen or an endogeneous antigen with MHC in antigen presenting cells (APC). It means the process of cutting.

As used herein, the term "major Histocompatibility Complex (MHC)" molecule is bound to the T cell T cell receptor (T cell) when the antigen presenting cell presents the processed antigen to the T cell. molecules recognized by receptors).

In the present specification, the "major histocompatibility complex" is abbreviated as "MHC" or "MHC molecule" when necessary. MHC molecules are broadly classified into class I MHC molecules and class II MHC molecules.

According to another preferred embodiment of the present invention, the bacterin III increases antigen presentation level through class I or class II major histocompatibility complex (MHC) in antigen presenting cells.

According to another preferred embodiment of the present invention, the bacterin III increases the antigen presenting level of internal antigen through the class I major histocompatibility complex (MHC) in antigen presenting cells.

According to another preferred embodiment of the present invention, the bacterin III increases the antigen presentation level of the external antigen through the class I or class II major histocompatibility complex (MHC) in the antigen presenting cells.

According to another preferred embodiment of the present invention, Bacatin III of the present invention improves the expression level of surface molecule, B7-1, CD40 or CD54 in antigen presenting cells.

According to another preferred embodiment of the invention, Bacatin III enhances the induction of antigen specific Cytotoxic T Lymphocyte (CTL) responses.

The active ingredient baccatin III of the present invention improves antigen-presenting ability through MHC molecules of antigen-presenting cells and increases antigen-induced ability to induce specific cytotoxic T Lymphocyte (CTL) response, thereby eventually enhancing immunity.

On the other hand, the bacterin III of the present invention enhances antigen presentation ability through an increase in the processing of antigens in antigen presenting cells, so that it can be labeled by a molecule recognized as an antigen by an immune response process in the body. It can be very usefully used for the treatment or prevention of disease. In the present invention, a disease that may be labeled by a molecule recognized as an antigen in an immune response process in the body is, for example, a viral infection disease or a bacterial infection disease, but is not limited thereto.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier in addition to the active ingredient Bacatin III. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the oral dosage of the pharmaceutical composition of the present invention is preferably 0.001-100 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, may be administered by intravenous infusion, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like. In the pharmaceutical composition of the present invention, the route of administration is preferably determined depending on the type of disease to which it is applied.

The concentration of the naphthalene derivative compound which is an active ingredient included in the composition of the present invention may be determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, and the like, and is not limited to a specific range of concentration.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The present invention relates to a pharmaceutical composition for immuno-enhancement comprising bacatin III as an active ingredient. The active ingredient Bacatin III of the composition of the present invention promotes the processing of antigen in Antigen Presenting Cell (APC), greatly increases the level of antigen presentation through the Major Histocompatibility Complex (MHC), Has activity to enhance the induction of specific Cytotoxic T Lymphocyte (CTL) responses. Therefore, the bacterin III of the present invention is very likely to be developed as an immunotherapeutic agent having immunopotentiating activity.

1 shows the results of increasing the antigen presenting ability of APC (Antigen Presenting Cell) of BM-DC. BM-DCs were incubated for 4 hours with the indicated amounts of soluble forms of Bacatin III or paclitaxel, followed by the addition of PLAG-nanoparticles (OVA / ov as 50 μg / ml) with OVA (ovalbumin). After incubation for 2 hours, cells were washed, fixed with paraformaldehyde, and evaluated for the ability to activate OVA (ovalbumin) -specific CD8 T cells using CD8 OVA1.3 cells (Panel A). ), DOBW cells were used to assess the ability to activate OVA-specific CD4 T cells (Panel B). Bacatin III or paclitaxel in the amounts indicated in culture of BM-DCs infected with rVV-OVA (MOI 5) were added. After 6 hours, dendritic cells (DCs) were washed, paraformaldehyde fixed, and then the ability to activate OVA-specific CD8 T cells using CD8 OVA1.3 cells was evaluated (Panel C). Each data point represents the mean SD SD from each of 3-5 experiments repeated three times. * p <0.05, ** p <0.01 vs. Bacatin III-untreated control.
2 shows that bacatin III does not affect the phagocytic activity of BM-DC. Bacatin III (1000 nM, Panel A) and paclitaxel (1000 nM, Panel B) were added to BM-DC for 4 hours. BM-DCs were then incubated with nanoparticles containing both OVA and FITC (NP [OVA + FITC]) for 2 hours, washed, recovered and analyzed by flow cytometry. Figure 2 shows a representative set of histograms of three individual experiments. Gray histograms show phagocytic activity of dendritic cells (DC) in the absence of bacterin III or paclitaxel, and thick solid histograms show phagocytic activity of dendritic cells (DC) in the presence of bacterin III or paclitaxel. . Dendritic cells (DCs) that were not incubated with FITC-labeled microspheres are shown as dotted histograms (left).
FIG. 3 shows that Bacatin III increases APC ability of dendritic cells (DCs) to present class I-restricted OVA peptides in vivo.
Panel A: DC 2.4 cells were incubated for 2 hours with nanoparticles containing only OVA (NP [OVA]) or nanoparticles containing both OVA and Bacatin III (NP [OVA + Bac-III]). Cells were then washed, paraformaldehyde fixed and the ability to activate OVA-specific CD8 T cells was evaluated using CD8 OVA1.3 cells. Each data point represents the mean &lt; RTI ID = 0.0 &gt; SD &lt; / RTI &gt; value obtained from each of three separate experiments performed three times. ** p <0.01 vs. Bacatin III-untreated control.
Panel B: Mice were injected with NP [OVA] or NP [OVA + Bacatin III] (200 μg iv as OVA per mouse) (n = 5 for each group). After 40 hours, dendritic cells were isolated from lymph nodes and spleen of mice using a negative selection method. The amount of class I MHC-restricted OVA (ovalbumin) peptide complex on dendritic cells was assessed using B3Z cells. B3Z cell responses were measured by X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside) staining. Each data point represents the mean square SD of values obtained from two representative experiments. ** p <0.01 vs. Bacatin III-untreated control.
4 shows the results of increased expression of OVA [257-264] -H-2K ^b ?? complexes. Mice were injected with NP [OVA] or NP [OVA + bacatin III] (200 μg OVA as OVA per mouse). After 40 hours, dendritic cells were isolated from lymph nodes of mice using the negative selection method. Expression levels of H-2K ^b molecules (Panel A) and OVA [257-264] -H-2K ^b complex (Panel B) were measured using monoclonal antibodies. Representative sets of histograms from five separate experiments are shown. Gray histograms are dendritic cells fed NP [OVA], and thick solid histograms represent dendritic cells fed NP [OVA + Bacatin III]. Dashed line represents isotype control.
FIG. 5 shows the results of measuring the effect of Bacatin III on the expression of class II MHC molecules and other cell surface molecules. Mice were injected with NP [OVA] or NP [OVA + bacatinIII] (200 μg OVA as OVA per mouse). After 40 hours, dendritic cells were isolated from lymph nodes and spleen of mice using the negative selection method. Expression of class II MHC molecules, adhesion molecules, and costimulatory molecules was measured using monoclonal antibodies. Representative sets of histograms from three separate experiments are shown. Gray histograms are dendritic cells fed NP [OVA], and thick solid histograms represent dendritic cells fed NP [OVA + baccatin III]. Dashed line represents isotype control.
FIG. 6 shows the results of efficient induction of CTL activity in mice by bacatin-exposed APC. Mice were injected with NP [OVA] or NP [OVA + bacatin III] (iv iv 100 μg OVA per mouse). After 5 days, in vivo CTL analysis was performed. Target cells used a 1: 1 mixture of syngeneic cells pulsed with OVA [257-264] peptide and labeled with high concentrations of CFSE and low concentrations of CFSE labeled without pulsing. . Target cells were injected (iv) into recipient mice and specific cytotoxicity was measured 18 hours later. Panel A shows representative histograms of lymph node cells from individual mice. Panel B graphically depicts the percentage of death of OVA [257-264] peptide-pulsed target cells in the spleen and lymph nodes. Each data point represents the mean SD SD of values from three separate experiments. ** p <0.01 vs. Bacatin III-untreated control.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

1. Cell Lines and Reagents

T cell hybridomas, B3Z86 / 90.14 (B3Z) (University of California, Berkeley, Calif.) And T cell hybridomas, CD8 OVA1.3 and DOBW (Case Western Reserve University, Cleveland, OH) Used [16-18]. The monoclonal antibody, Alexa 488 conjugated 25-D1.16, which recognizes recombinant Bexonia virus (rVV-OVA) and OVA [257-264] -K ^b complexes encoding OVA (ovalbumin), has been described in National Institutes of Health, Bethesda. , MD from [19]. Dendritic cells derived from bone marrow (BM-DC) were generated from bone marrow progenitor cells by treating GM-CSF as already established [10]. Dendritic cell line, DC2.4, was purchased from Dana-Farber Cancer Institute (Harvard Medical School, Boston, Mass.) [20]. Paclitaxel and baccatin III (Sigma-Aldrich St. Louis, Mo.) were dissolved in ethanol and ethanol / Tween-20 / PBS (10/10/80) and used for in vitro and in vivo experiments, respectively. It was.

2. Preparation of PLGA Nanoparticles

Nanoparticles containing OVA (NP [OVA]) were prepared using a homogenization / solvent evaporation method. Prepared using 400 μl of OVA-containing water (50 mg OVA / ml) and 2 ml of ethyl acetate containing poly (lactic-co-glycolic acid) (100 mg / ml, Sigma-Aldrich) [21] ]. Bacatin III or fluorescein isothiocyanate (FITC) was added to the ethyl acetate with PLGA and incorporated into PLGA-nanoparticles. OVA content was measured using a micro-bicinchoninic acid assay kit (Pierce, Rockford, IL) after dissolving nanoparticles in lysis buffer containing 0.1% SDS and 0.1N NaOH. The baccatin III content was measured by high performance liquid chromatography (HPLC) using a Nucleosil C18 column (2.5 × 250 mm, 10 μm) after the nanoparticles were dissolved in methanol.

3. MHC Class I-Limited T Cell Activation Assay

In vitro analysis of MHC class I-limited T cell activation was performed according to known methods using CD8 OVA1.3 cells that recognize OVA [257-264] -K ^b complex and secrete IL-2 [ 17]. Briefly, Bacatin III was added to a culture plate of BM-DCs (1 × 10 ⁵ cells / well) generated from bone marrow cells of C57BL / 6 (H-2b) mice. Plates were incubated at 37 ° C. for 4 hours, then NP [OVA] (50 μg OVA / mL) was added. After incubation at 37 ° C. for 2 hours, the plates were washed twice with 300 μl of pre-warmed PBS per well and 5 at room temperature with 100 μl of 1.0% paraformaldehyde per well cooled with ice. Fixed for a minute. Plates were washed three times with 300 μl PBS per well. Then CD8 OVA1.3 cells were added (2 × 10 ⁵ cells / well). After incubation at 37 ° C. for 18 hours, the plates were centrifuged at 400 g, the culture supernatants were recovered and analyzed for IL-2 content using the IL-2 ELISA Kit (BD Biosciences).

4. MHC Class II-Limited T Cell Activation Assay

In vitro assays for MHC class II-limited T cells were performed according to known methods using DOBW cells recognizing OVA [323-339] -IA ^d complex and secreting IL-2 [18]. Briefly, Bacatin III was added to a culture plate of BM-DCs (1 × 10 ⁵ cells / well) generated from bone marrow cells of BALB / c (H-2 ^d ) mice. Plates were then incubated at 37 ° C. for 4 hours and NP [OVA] (50 μg OVA / mL) was added. After incubation at 37 ° C. for 2 hours, the plate was fixed with 1.0% paraformaldehyde by the method as described in section 3. above. After washing the plates with PBS, DOBW cells (1 × 10 ⁵ cells / well) were added and the plates were incubated again at 37 ° C. for 48 hours. IL-2 content in the culture supernatant was analyzed using the IL-2 ELISA kit (BD Biosciences).

5. Viral Antigen Presentation Assay

Dendritic cells (1 × 10 ⁷ / ml) were infected with rVV-OVA (multiplicity of infection, 5) at 37 ° C. for 20 minutes. Virus-infected dendritic cells (DCs) were diluted to 1 × 10 ⁶ cells / ml using DMEM containing 10% FBS and 1 μM cytosine β-D-arabinofuranoside (Sigma-Aldrich) and 96 wells Dispense into microtiter plates (100 μl / well). After addition of the indicated amount of Bacatin III, the plates were incubated at 37 ° C. for 6 hours, washed twice with pre-warmed PBS in an amount of 300 μl per well, and ice-cooled 1.0% paraformaldehyde. Was used in an amount of 100 μl per well and fixed at room temperature for 5 minutes. Plates were washed three times with 300 μl PBS per well, CD8 OVA 1.3 cells (2 × 10 ⁵ / well) were added and plates were incubated at 37 ° C. for 24 hours. The culture supernatant was recovered after centrifugation at 400 g, and the IL-2 content was measured using the IL-2 ELISA kit (BD Biosciences).

6. In vivo cross-presentation assay

C57BL / 6 mice were immunized (iv) using OVA-comprising nanoparticles or nanoparticles comprising both OVA and Bacatin III. After 24 hours, mouse dendritic cells were prepared from single cell suspensions prepared in cell pools of spleen, inguinal, mesenteric and axillary lymph nodes or in a pool of spleen collected from at least 10 mice per treatment group. Dendritic cells were isolated using a mouse DC enrichment kit (StemSep, Vancouver, Canada) [11]. MHC class I-limited T cell activation assays were performed according to known methods using B3Z cells expressing β-galactosidase when activated by OVA [257-264] -K ^b complex [11, 16]. ]. B3Z cell responses were measured by X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside) staining [11].

7. Phagocytosis Activity

Nanoparticles containing both OVA and FITC were added to dendritic cells. After 20 minutes, nanoparticles not absorbed by phagocytosis were removed by washing with pre-warmed PBS. Cells were collected, fixed in 1.0% paraformaldehyde in PBS, and flow cytometry using a FACSCanto flow cytometer (BD Biosciences).

8. Phenotype analysis

As described previously, cells were stained with monoclonal antibodies that recognize murine cell surface markers [22] and flow cytometry was performed. Monoclonal antibody, anti-H2-Kb antibody, anti-I-Ab antibody, anti-CD80 antibody, anti-CD86 antibody, anti-CD40 antibody, anti-CD54 antibody and isotype-matched control antibody antibody) was purchased from BD Biosciences.

9. In vivo CTL Analysis

C57BL / 6 mice were immunized (iv) with 100 μg NP [OVA] or NP [OVA + Bacatin III]. After 7 days, cytotoxic phosphorus using CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester, Molecular Probes) -labeled target cells prepared from syngeneic spleen and lymph node cell suspensions, according to already known methods. Vivo evaluation was performed [11]. In vivo specific cytotoxicity was determined by flow cytometry on lymph nodes and splenocytes isolated from recipient mice 18 hours after iv injection. Non-coated vs. The ratio between the percentages of OVA [257-264] -coating (CFSE ^low / CFSE ^high ) was calculated to obtain arithmetic values for cytotoxicity.

10. Statistical Analysis

Statistical significance between control and treatment groups was assessed using one-way ANOVA and Student's t-test.

Experiment result

1. Baccatin III increases the APC ability of OVA-feeding BM-DCs against OVA-specific T cells.

To determine the effect of baccatin III on the function of APC (Antigen Presenting Cell) on MHC class I-restricted T cells, from bone marrow cells of C57BL / 6 mice (H- ^2b ) BM-DCs produced by GM-CSF treatment were treated with Bacatin III for 4 hours and phagocytose for 2 hours by addition of nanoparticles containing OVA (NP [OVA]). BM-DCs were then fixed with paraformaldehyde, and the APC function of the fixed cells was evaluated using OVA-specific CD8 T cell hybridoma CD8 OVA1.3 cells. As shown in panel A of FIG. 1, dendritic cells (DCs) increased IL-2 secretion from CD8 OVA1.3 cells when treated with baccatin III.

The effect of Bacatin III on the function of APC (Antigen Presenting Cell) on MHC class II-restricted T cells was generated by GM-CSF treatment from bone marrow cells of BALB / C mice (H-2 ^d ) Was measured using BM-DC. BM-DCs were treated with Bacatin III for 4 hours and phagocytose for 2 hours by addition of nanoparticles containing OVA (NP [OVA]). Subsequently, BM-DC was fixed with paraformaldehyde, and the APC function of the fixed dendritic cells was evaluated using OVA-specific CD4 T cell hybridoma DOBW cells. As shown in panel B of FIG. 1, dendritic cells (DCs) treated with bacatin III increased the secretion of IL-2 from DOBW cells.

The effect of Bacatin III on the MHC class I-limited presentation of endogenous antigens was achieved using dendritic cells (DCs) infected with a recombinant Vaccinia virus encoding OVA (rVV-OVA). Was investigated. In this experiment, Bacatin III was added to the culture of BM-DCs infected with rVV-OVA, and after 6 hours dendritic cells (DCs) were fixed in paraformaldehyde, and the APC function of the fixed dendritic cells was OVA-specific. Red CD8 T cells were evaluated using hybridoma CD8 OVA1.3 cells. As shown in panel C of FIG. 1, treatment of rVV-OVA infected dendritic cells (DCs) with baccatin III increased IL-2 secretion from CD8 OVA1.3 cells.

Taxol, a clinical anticancer agent chemically synthesized from Bacatin III, showed no activity as seen in Bacatin III. Rather, Taxol inhibited the secretion of IL-2 from OVA-specific CD4 T cells and OVA-specific CD4 T cells upon high concentration treatment.

2. Bacatin III does not affect the phagocytic activity of dendritic cells.

To determine whether the activity of increasing bacterium III antigen presentation was due to an increase in phagocytic activity, dendritic cells (DCs) were treated with different concentrations of baccatin III or taxol for 4 hours and included both OVA and FITC. Incubated for 2 hours with nanoparticles. Subsequently, the dendritic cells were washed with ice-cold PBS and collected by weak pipetting. As a result of flow cytometry on the recovered cells, Bacatin III had no effect on phagocytic activity of dendritic cells, while taxol had a significant effect on phagocytosis of dendritic cells. 2 shows the experimental results.

3. Bacatin III increases class I-restricted OVA peptide presentation in vivo.

To deliver Bacatin III to phagocytes in vivo, nanoparticles comprising Bacatin III and OVA were prepared using biodegradable / biocompatible polymer PLGA. The amount of Bacatin III contained in the nanoparticles was measured by high performance liquid chromatography (HPLC) after degrading the nanoparticles in methanol, and as a result, it was determined that Bacatin III contained 52 μg per mg OVA. The effect of the inclusion of bacatin III in the nanoparticles was measured using DC cell line DC2.4 cells [20]. As shown in panel A of FIG. 3, the nanoparticles (NP [OVA + Bac-III]) containing both OVA and Bacatin III had CD8 OVA1. Compared with nanoparticles containing only OVA (NP [OVA]). The secretion of IL-2 from 3 cells was induced more.

To test for in vivo relevance, nanoparticles containing only OVA or nanoparticles containing both OVA and baccatin III were injected intravenously into the tail vein of the mouse (200 μg OVA per mouse). After 40 hours, dendritic cells were isolated from lymph nodes and spleen of mice using a negative selection method, and the efficiency of class I MHC-restricted OVA peptide presentation was determined using OVA-specific CD8 T hybridoma B3Z cells. It evaluated using. When Baccarmin III was added to OVA-nanoparticles, B3Z cell activation of splenic dendritic cells and lymph node dendritic cells was greatly improved (Panel B of FIG. 3). B3Z cells express β-galactosidase when stimulated with only the OVA [257-264] -K ^b complex, the recognized 'signal 1' required for T cell activation [16], thereby expressing β-galactosidase The level may reflect the amount of OVA [257-264] -K ^b complex on dendritic cells.

To provide additional evidence that Bacatin III increases MHC class I-restricted OVA peptide presentation in dendritic cells in vivo, dendritic cells (DCs) isolated from lymph nodes were treated with OVA [257-264] -K ^b. Stained with F (ab ') ² fragment of 25-D1.16 -specific monoclonal antibody [19]. As can be seen in panel A of FIG. 4, the addition of Bacatin III to OVA-nanoparticles did not affect the total level of H-2K ^b . However, the addition of Bacatin III to OVA-nanoparticles increased the expression of the OVA [257-264] -H-2K ^b complex to discernible levels.

4. Effect of Bacatin III on the Expression of Class II MHC Molecules and Other Cell Surface Molecules

Effects of Bacatin III on the Expression of Cell Surface Molecules Using Dendritic Cells (DCs) Isolated from Lymph Nodes of Intravenously Injected Nanoparticles Containing OVA or Nanoparticles Containing Both OVA and Bacatin III Inspected. Bacatin III increased B7-1, CD40 and CD54 expression levels to discernible levels, while having negligible effects on the expression of class II MHC and B7-2 molecules (see FIG. 5).

5. Bacatin III-exposed antigen presenting cells (APCs) effectively induce CTL responses in vivo.

To determine whether the increase in MHC-restricted antigen presentation is functionally related to the induction of antigen-specific T cell responses, nanoparticles containing only OVA (NP [OVA]) and nanoparticles containing both baccatin III and OVA The CTL inducing activity of the particles (NP [OVA + Bac-III]) was compared with each other. In this experiment, nanoparticles were injected intravenously through the tail vein of mice (100 μg OVA per mouse). After 5 days, in vivo CTL analysis was performed using CFSE® labeled syngeneic target cells in mice immunized with nanoparticles. Panel A of FIG. 6 is a representative histogram. Addition of Bacatin III to OVA-nanoparticles greatly increased the ability to induce OVA-specific CTL in the spleen and lymph nodes (see panel B of FIG. 6).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (6)

(a) a pharmaceutically effective amount of baccatin III; And (b) a pharmaceutically acceptable carrier.
According to claim 1, wherein the bacterin III composition characterized in that to increase the level of antigen presentation through the major histocompatibility complex (MHC) in the Antigen Presenting Cell (APC).
3. The composition of claim 2, wherein the baccatin III increases the antigen presenting level of the internal antigen via class I major histocompatibility complex (MHC).
3. The composition of claim 2, wherein the baccatin III increases the antigen presenting level of an external antigen via a class I or class II major histocompatibility complex (MHC).
According to claim 1, wherein the bacterin III composition characterized in that to enhance the expression level of B7-1, CD40 or CD54 which is the surface molecule of the antigen-presenting cells.
According to claim 1, wherein the bacterin III composition characterized in that to enhance the ability to induce antigen-specific Cytotoxic T Lymphocyte (CTL) response.

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